Reduced risk tobacco products and methods of making same

ABSTRACT

Embodiments provided herein concern tobacco and tobacco products having a reduced amount of a harmful compound. More specifically, several embodiments concern approaches to modify the expression of a gene that is involved in the production of a harmful compound in tobacco, tobacco products made using these approaches and methods of determining whether the removal of said compounds using said approaches yields a tobacco and/or a tobacco product that has a reduced potential to contribute to a tobacco-related disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/243,675, filed Aug. 22, 2016, which is a continuation of U.S.application Ser. No. 14/102,340, filed Dec. 10, 2013, now U.S. Pat. No.9,439,452, which is a continuation of U.S. application Ser. No.11/913,870, filed Mar. 25, 2011, now abandoned, which is a 371 entryinto the U.S. of Paris Convention Application No. PCT/US2006/018065,filed May 10, 2006, which claims the benefit of priority to U.S.Provisional Application Ser. No. 60/680,283, filed May 11, 2005. Each ofthe above-mentioned priority documents is incorporated by reference inits entirety into the present application.

FIELD OF THE INVENTION

The invention relates to reduced risk tobacco and tobacco products andmethods for detecting, identifying and evaluating such tobacco andtobacco products to determine the potential that these compositions haveto contribute to a tobacco-related disease.

BACKGROUND

The leading preventable cause of death and disability in the UnitedStates is the chronic use of tobacco products, in particular,cigarettes. In addition to lung cancers, tobacco use plays importantdirect and indirect roles in the etiology of a wide range of othercancers, including those of the upper aerodigestive tract (i.e., oralcavity, pharynx, larynx, and esophagus), bladder, stomach, kidney,pancreas, uterine cervix, and blood (myeloid leukemia). Exposure totobacco carcinogens and toxins is also a major cause of other diseasesof the pulmonary system (e.g., bronchitis, emphysema, and chronicobstructive pulmonary disease), the cardiovascular system (e.g., stroke,atherosclerosis, and myocardial infarction), and the female reproductivesystem (e.g., increased risk of miscarriage, premature delivery, lowbirth weight, and stillbirth). While numerous studies have elucidatedsome of the biological effects of cigarette smoke that result in itsability to induce this range of pathologies in smokers, little is knownabout the nature and temporal association of molecular events that drivespecific stages in the multi-step processes that result in clinicallyevident disease. This is due to the fact that cigarette smoke is acomplex chemical mixture of gases and suspended particulate materialthat consists of a wide variety of condensed organic compounds (i.e.,‘tar’) that collectively contain a large number of toxins, carcinogens,co-carcinogens, mutagens, and reactive organic and inorganic molecules.Thus, there is a pressing need to decrease the health risk caused bytobacco products.

SUMMARY

Embodiments described herein generally relate to tobacco and/or tobaccoproducts having a reduced amount of a harmful compound, and methods ofdeveloping, screening and using such tobacco and tobacco products. Forexample, several approaches are provided to reduce the amount of one ormore harmful compounds in tobacco by, for example, modifying theexpression of a gene that is involved in the production of a harmfulcompound in tobacco. Also provided are methods of determining whetherthe removal of a harmful compound yields a tobacco and/or a tobaccoproduct that has a reduced potential to contribute to a tobacco-relateddisease. Also provided are reduced-risk tobacco and tobacco productsmade in accordance with the methods provided herein. Also provided aremethods of using the reduced-risk tobacco and tobacco products made inaccordance with the methods provided herein.

As described in more detail below, provided herein are nucleic acidmolecules and nucleic acid constructs that contain sequences that can beused to inhibit expression of a gene involved in the biosynthesis of acompound associated with a tobacco-related disease. Also provided hereinare modified tobaccos and modified tobacco products that have beenmodified by composition and/or configuration in order to deliver to theuser a reduced amount of a compound associated with a tobacco-relateddisease. Exemplary modified tobaccos are tobaccos that have beengenetically modified to contain a reduced amount of a compoundassociated with a tobacco-related disease. Exemplary geneticallymodified tobaccos are those containing the nucleic acid molecules orconstructs provided herein. Exemplary modified tobacco products arethose containing modified tobacco or, those containing a modifiedfilter, where the modification results in delivery to the user of areduced amount of a compound associated with a tobacco-related disease.

Also provided herein are methods of analyzing tobacco products such asthe modified tobacco and modified tobacco products described herein, soas to determine whether the tobacco product is a reduced risk product(e.g., a product that has a reduced propensity to modulate cellularhomeostasis, or a reduced level of induction of a cellular marker for atobacco-related disease). Some of these methods can be practiced, forexample, by identifying a compound that is related to a tobacco-relateddisease (e.g., nicotine or a sterol), removing the compound or aprecursor for the compound by modification to the tobacco or tobaccoproduct, and analyzing the ability of the modified tobacco or modifiedtobacco product to contribute to a tobacco related disease by monitoringthe impact of the modified tobacco or modified tobacco product on amarker for cellular homeostasis. In one example, a cellular marker for atobacco related disease is monitored. In another example, thetranscriptome and/or proteome of the cell is monitored. These methodscan be used for both in vitro and in vivo testing. That is, the samecellular markers that have been identified in the in vitro studies canbe analyzed in smokers that consume reduced risk cigarettes developedaccording to the methods above and this data can be compared to theimpact on the same cellular markers in smokers that consume conventionalcigarettes. By these approaches, a cigarette that minimizes thedisruptions of the cellular environment of a smoker can be obtained.

Further provided herein are kits that contain the modified tobacco ormodified tobacco products provided herein, and smoking cessationprograms, which utilize the modified tobacco or modified tobaccoproducts provided herein.

Provided herein are methods of making a tobacco product with a reducedpotential to contribute to a tobacco related disease by providing agenetically modified tobacco configured to deliver a reduced amount of acompound that contributes to a tobacco related disease, as compared to areference tobacco or a conventional tobacco, contacting a mammalian cellwith smoke, or a smoke condensate obtained from said geneticallymodified tobacco, identifying a modulation of homeostasis of said cell,as compared to a control cell, which has been contacted with smoke, or asmoke condensate obtained from said reference tobacco or saidconventional tobacco, wherein a decreased modulation of homeostasis insaid cell compared to modulation of homeostasis in said control cellindicates a reduction in the potential to contribute to a tobaccorelated disease, and incorporating said identified genetically modifiedtobacco into a tobacco product. In some such methods, modulation ofhomeostasis in the cell is identified by determining the presence,absence or level of a molecular marker in the cell. In some suchmethods, the mammalian cell is a lung cell or a cell of the oral cavity.In some such methods, the genetically modified tobacco is identified asproducing a reduced amount of a compound that contributes to a tobaccorelated disease, as compared to a conventional tobacco product of thesame class or a reference tobacco product of the same class. In somesuch methods, the genetically modified tobacco is incorporated into atobacco product that contains a filter, which retains an increasedamount of a compound that contributes to a tobacco related disease, ascompared to a reference filter or a conventional filter. In some suchmethods, the genetically modified tobacco comprises a heterologousnucleic acid that inhibits expression of an enzyme in the nicotinebiosynthetic pathway. In some such methods, the heterologous nucleicacid inhibits expression of at least two enzymes in the nicotinebiosynthetic pathway. In some such methods, the genetically modifiedtobacco comprises a heterologous nucleic acid that inhibits expressionof an enzyme in the sterol biosynthetic pathway. In some such methods,the heterologous nucleic acid inhibits expression of at least twoenzymes in the sterol biosynthetic pathway. In some such methods, thegenetically modified tobacco comprises a heterologous nucleic acid thatinhibits expression of an enzyme in the nicotine biosynthetic pathwayand an enzyme in the sterol biosynthetic pathway. In some such methods,the genetically modified tobacco has a reduced amount of nornicotine anda conventional amount of nicotine. In some such methods, geneticallymodified tobacco comprises a nucleic acid construct selected from thegroup consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49 and 50. Also provided herein are tobacco products made by the methodprovided herein.

Also provided herein are tobacco products comprising a geneticallymodified tobacco that comprises a reduced amount of nicotine as comparedto a conventional tobacco product of the same class or a referencetobacco product of the same class and a heterologous nucleic acid thatinhibits expression of at least two enzymes involved in nicotinebiosynthesis. Also provided herein are tobacco products comprising agenetically modified tobacco that comprises a reduced amount of a sterolas compared to a conventional tobacco product of the same class or areference tobacco product of the same class and a heterologous nucleicacid that inhibits expression of an enzyme involved in sterolbiosynthesis. In some such tobacco products, the genetically modifiedtobacco comprises a nucleic acid construct as described herein. In somesuch tobacco products, the genetically modified tobacco comprises anucleic acid construct selected from the group consisting of SEQ. ID.NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50.

In the methods and tobacco products provided herein, the geneticallymodified tobacco comprises a reduced activity of a gene selected fromthe group consisting of arginine decarboxylase (ADC), methylputrescineoxidase (MPO), NADH dehydrogenase, omithine decarboxylase (ODC),phosphoribosylanthranilate isomerase (PRAI), putrescineN-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT),S-adenosyl-methionine synthetase (SAMS), or A622 or comprises aninhibition of a gene that regulates the production of sterolbiosynthesis include HMG-CoA reductase, 14alpha demethylase, squalenesynthase, SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase, andC4-demethylase. In the methods and tobacco products provided herein, thegenetically modified tobacco has reduced production of a compound thatcontributes to a tobacco related disease which is stable over at least2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40 or 50 generations. In themethods and tobacco products provided herein, the genetically modifiedtobacco has agronomic characteristics suitable for commercialproduction. In the methods and tobacco products provided herein, theagronomic characteristics are phenotypically different from conventionaltobacco, and said agronomic characteristics can be compensated for byconventional agronomic methods. In the methods and tobacco productsprovided herein, the conventional agronomic methods are selected fromthe group consisting of irrigation, administration of fertilizer, andadministration of nutrients.

Also provided herein are genetically modified tobaccos that produce areduced amount of a compound that contributes to a tobacco relateddisease, as compared to a conventional tobacco product of the same classor a reference tobacco product of the same class, comprising aheterologous nucleic acid that inhibits expression of an enzyme in thebiosynthetic pathway of a compound that contributes to a tobacco relateddisease. Also provided herein are reduced risk tobacco productscomprising a genetically modified tobacco that produces a reduced amountof a compound that contributes to a tobacco related disease, as comparedto a conventional tobacco product of the same class or a referencetobacco product of the same class. In some such tobaccos or tobaccoproducts, the modified tobacco comprises a nucleic acid construct asdescribed herein. In some such tobaccos or tobacco products, themodified tobacco comprises a heterologous nucleic acid that inhibitsexpression of at least two enzymes in the nicotine biosynthetic pathway.In some such tobaccos or tobacco products, the modified tobaccocomprises a heterologous nucleic acid that inhibits expression of atleast two enzymes in the sterol biosynthetic pathway.

Also provided herein are methods of making a reduced risk tobaccoproduct by providing a modified tobacco or modified tobacco productconfigured to deliver to a user a reduced amount of a compound thatcontributes to a tobacco related disease, as compared to a referencetobacco or tobacco product or a conventional tobacco or tobacco product,contacting smoke or smoke condensate obtained from said modified tobaccoor modified tobacco product with a cell, identifying a modulation ofhomeostasis of said cell, as compared to a control cell, which has beencontacted with smoke or a smoke condensate obtained from said referencetobacco or tobacco product or said conventional tobacco or tobaccoproduct, wherein a decreased modulation of homeostasis in said cellcompared to modulation of homeostasis in said control cell indicates areduction in the potential to contribute to a tobacco related disease,and incorporating said modified tobacco or modified tobacco product intosaid reduced risk tobacco product. In some such methods, modulation ofhomeostasis in the cell is identified by determining the presence,absence or level of a molecular marker in the cell. In some suchmethods, the modified tobacco is genetically modified tobacco. In somesuch methods, the genetically modified tobacco is modified according tothe methods provided herein.

Also provided are reduced risk tobaccos as substantially describedherein. Also provided are reduced risk tobacco products as substantiallydescribed herein. Also provided are uses of the tobaccos or tobaccoproducts provided herein.

Also provided are isolated nucleic acids substantially as describedherein. Also provided are isolated inhibition cassettes substantially asdescribed herein.

Also provided is a genetically modified tobacco having a reduced amountof nicotine as compared to conventional tobacco, further comprising aheterologous nucleic acid that encodes a gene that produces acomposition selected from the group consisting of a medicinal compound,industrial oil, or dietary supplement, wherein said composition issubstantially not present in conventional or wild-type tobacco. In somesuch tobaccos, the medicinal compound is an antibody or fragment thereofor an immunogenic preparation. In some such tobaccos, the medicinalcompound is a vaccine preparation. In some such tobaccos, the medicinalcompound is a veterinary product.

Also provided are genetically modified tobaccos that produce a reducedamount of a compound that contributes to a tobacco related disease, ascompared to a conventional tobacco product of the same class or areference tobacco product of the same class, comprising a heterologousnucleic acid that inhibits expression of an enzyme in the biosyntheticpathway of a compound that contributes to a tobacco related disease.Also provided are reduced risk tobacco products comprising a geneticallymodified tobacco that produces a reduced amount of a compound thatcontributes to a tobacco related disease, as compared to a conventionaltobacco product of the same class or a reference tobacco product of thesame class. In some such tobaccos or tobacco products, the compound isnicotine. In some such tobaccos or tobacco products, the compound is asterol. In some such tobaccos or tobacco products, the compound is aTSNA. In some such tobaccos or tobacco products, the compound is a PAH.In some such tobaccos or tobacco products, the compound is nornicotine.In some such tobaccos or tobacco products, the genetically modifiedtobacco has a reduced amount of nornicotine and a conventional amount ofnicotine. In some such tobaccos or tobacco products, the geneticallymodified tobacco comprises a nucleic acid construct as described herein.In some such tobaccos or tobacco products, the genetically modifiedtobacco comprises a nucleic acid construct selected from the groupconsisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and50. In some such tobaccos or tobacco products, expression of two or moregenes in the biosynthetic pathway of said compound is inhibited. In somesuch tobaccos or tobacco products, the genetically modified tobaccocomprises two or more nucleic acid constructs as described herein. Insome such tobaccos or tobacco products, the genetically modified tobaccocomprises two or more nucleic acid constructs selected from the groupconsisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and50. Some such tobaccos or tobacco products comprise reduced activity ofa gene selected from the group consisting of arginine decarboxylase(ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, omithinedecarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI),putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), or A622 orcomprises an inhibition of a gene that regulates the production ofsterol biosynthesis include HMG-CoA reductase, 14alpha demethylase,squalene synthase, SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase,and C4-demethylase. Some such tobaccos or tobacco products comprise agenetically modified tobacco for which reduced production of a compoundthat contributes to a tobacco related disease is stable over at least 2,3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40 or 50 generations. Some suchtobaccos or tobacco products comprise a genetically modified tobaccohaving agronomic characteristics suitable for commercial production.

Also provided herein are methods of making a reduced risk tobaccoproduct by providing a modified tobacco or modified tobacco productconfigured to deliver to a user a reduced amount of a compound thatcontributes to a tobacco related disease, as compared to a referencetobacco or tobacco product or a conventional tobacco or tobacco product,contacting smoke or smoke condensate obtained from said modified tobaccoor modified tobacco product with a cell identifying a modulation ofhomeostasis of said cell, as compared to a control cell, which has beencontacted with smoke or a smoke condensate obtained from said referencetobacco or tobacco product or said conventional tobacco or tobaccoproduct, wherein a decreased modulation of homeostasis in said cellcompared to modulation of homeostasis in said control cell indicates areduction in the potential to contribute to a tobacco related disease,and incorporating said modified tobacco or modified tobacco product intosaid reduced risk tobacco product. In some such methods, modulation ofhomeostasis in the cell is identified by determining the presence,absence or level of a molecular marker in the cell. In some suchmethods, the modified tobacco is genetically modified tobacco. In somesuch methods, the genetically modified tobacco is modified according toany of methods provided herein. In some such methods, the geneticallymodified tobacco is identified as producing a reduced amount of acompound that contributes to a tobacco related disease, as compared to aconventional tobacco product of the same class or a reference tobaccoproduct of the same class. In some such methods, the modified tobaccoproduct contains a filter that retains an increased amount of a compoundthat contributes to a tobacco related disease, as compared to areference filter or a conventional filter. Also provided herein arereduced risk tobacco products made by any of the methods providedherein. Also provided herein are methods of using a reduced risk tobaccoproduct of any of the methods provided herein to reduce the potential ofan individual that smokes to acquire a tobacco related diseasecomprising identifying an individual in need of a reduced risk tobaccoproduct and providing the individual the tobacco product of the methodsprovided herein.

Also provided herein are plant cells resistant to norflurazonecomprising providing said cell the nucleic acid of SEQ ID No 10, 11, or12; and also provided herein are method of making the same.

Also provided herein are crops of plants comprising the nucleic acid ofSEQ ID No 10, 11, or 12. Also provided herein are methods of cultivationof a crop of plants comprising obtaining plants with the nucleic acid ofSEQ ID No 10, 11, or 12, cultivating said plants, and contacting saidplants with norflurazone.

Also provided herein are methods of selecting positively transformedplant cells comprising providing the nucleic acid of SEQ ID No 10, 11,or 12 to said plant cells and contacting said plant cells withnorflurazone, whereby the cells that survive contact with norflurazoneare positively transformed plant cells.

Also provided herein are isolated nucleic acids substantially asdescribed herein. Also provided herein are isolated inhibition cassettessubstantially as described herein. Also provided herein are isolatedselection cassettes substantially described herein, wherein saidselection cassette comprises the sequence of SEQ ID No 10, 11, or 12.Also provided herein are reduced risk tobaccos substantially describedherein. Also provided herein are reduced risk tobacco productssubstantially described herein.

Also provided herein are reduced risk tobacco products comprising atransgenic tobacco that comprises a reduced expression of a plurality ofgenes that regulate the production of at least two different compoundsin said tobacco that contribute to a tobacco related disease. In somesuch tobacco products, the two different compounds in said tobacco arenicotine and a sterol.

Also provided herein are kits comprising two or more different tobaccosor tobacco products in accordance with any of the methods providedherein. In some such kits, the different tobaccos or tobacco productsare differently labeled.

Also provided herein are uses of a tobacco or tobacco product of any ofthe methods, tobaccos, tobacco products or kits provided herein. Somesuch uses are tobacco-use cessation methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. An illustration of a QPTase inhibition construct comprising aQPTase inhibition cassette including full-length QPTase coding sequenceand a GUS selection cassette.

FIG. 2. An illustration of a QPTase inhibition construct comprising aQPTase inhibition cassette including a 360 bp fragment of the QPTasegene and a norflurazone resistance selection cassette including a mutantphytoene desaturase gene (PDSM-1).

FIG. 3. An illustration of a PMTase inhibition construct comprising aPMTase inhibition cassette including a 241 bp fragment of the PMTasegene and a norflurazone resistance selection cassette including a mutantphytoene desaturase gene (PDSM-1).

FIG. 4. An illustration of a A622 inhibition construct comprising a A622inhibition cassette including a 628 bp fragment of the A622 gene and anorflurazone resistance selection cassette including a mutant phytoenedesaturase gene (PDSM-1).

FIG. 5. An illustration of a QPTase/A622 double inhibition constructcomprising a QPTase/A622 inhibition cassette including a 360 bp fragmentof the QPTase gene and a 628 bp fragment of the A622 gene and anorflurazone resistance selection cassette including a mutant phytoenedesaturase gene (PDSM-1).

FIG. 6. An illustration of a SMT2/A622 double inhibition constructcomprising a A622 inhibition cassette including a 628 bp fragment of theA622 gene, an SMT2 inhibition cassette including a 779 bp fragment ofthe SMT2 gene and a norflurazone resistance selection cassette includinga mutant phytoene desaturase gene (PDSM-1).

FIG. 7. An illustration of a QPTase inhibition construct comprising aQPTase inhibition cassette including a 360 bp fragment of the QPTasegene and a norflurazone resistance selection cassette including a mutantphytoene desaturase gene (PDSM-1).

FIG. 8. An illustration of a QPTase inhibition construct comprising aQPTase inhibition cassette including a 360 bp fragment of the QPTasegene and a norflurazone resistance selection cassette including a mutantphytoene desaturase gene (PDSM-1).

FIG. 9. An illustration of a PMTase inhibition construct comprising aPMTase inhibition cassette including a 202 bp fragment of the PMTasegene and a norflurazone resistance selection cassette including a mutantphytoene desaturase gene (PDSM-1).

FIG. 10. An illustration of a PMTase inhibition construct comprising aPMTase inhibition cassette including a 344 bp fragment of the PMTasegene and a norflurazone resistance selection cassette including a mutantphytoene desaturase gene (PDSM-1).

FIG. 11. An illustration of a QPTase inhibition construct comprising aQPTase inhibition cassette including a 360 bp fragment of the QPTasegene and a kanamycin resistance selection cassette including a neomycinphosphotransferase gene (NPTII).

FIG. 12A-B. Fluorescence photomicrographs of NHBE cells exposed to 25μg/ml of tobacco smoke condensate for 24 h. The cells stained with DAPIand immuno-stained with γH2AX Ab were examined under UV light—(A) orblue light—(B) fluorescence excitation (Nikon Microphot FXA, 60×Objective.).

FIG. 13A-C. Bivariate (cellular DNA content vs cell immunofluorescence)distributions (scatterplots) of A549 cells, mock-treated (B) or exposedfor 30 min to tobacco smoke (A, C), immuno-stained either with γH2AX Ab(B,C) or with an isotype control IgG (A). The dashed-line represents themaximal fluorescence level (for 99% cells) of the IgG control.

FIG. 14. Plots showing the percent increase (Δ) in mean γH2AXimmunofluorescence of A549 cells (per unit of DNA) exposed to smoke fordifferent time intervals, calculated for cells in particular phases ofthe cell cycle, as described in Example 1. The value for mock-exposedcells was subtracted from those exposed to smoke.

FIG. 15. Plots showing percent increase (Δ) in mean γH2AXimmunofluorescence of NHBE cells treated with 10, 25 or 50 μg/mlconcentrations of smoke condensate for different periods of time. As inFIG. 14, the γH2AX value for the mock-exposed cells was subtracted fromthe values of the cells exposed to different concentrations ofcondensate.

FIG. 16. Percent increase (Δ) in mean γH2AX immunofluorescence of NHBEcells treated with 10 μg/ml of smoke condensate for different intervalsof time, in relation to cell cycle phase. As in FIG. 14, the γH2AX valuefor the mock-exposed cells was subtracted from the values of the cellsexposed to condensate.

FIG. 17. Plots showing the percent increase (Δ) in mean γH2AXimmunofluorescence of A549 cells (per unit of DNA) exposed to smoke ofIM16 cigarettes for different time intervals, calculated for cells inparticular phases of the cell cycle, as described in Example 2.

FIG. 18A-D. (A) Plots showing increase (Δ) in mean γH2AXimmunofluorescence of A549 cells exposed to smoke of IM16 cigarettes for15 minutes, relative to mock exposed cells. (B) Scatter plots showingthe increase in γH2AX following 60 min of recovery of the A549 cells inparticular phases of the cell cycle for mock exposed (upper plot) andfor IM16 smoke exposed (lower plot) cells. (C) Plots showing increase(Δ) in mean γH2AX immunofluorescence of NHBE cells exposed to smoke ofIM16 cigarettes for 20 minutes, relative to mock exposed cells. (D)Scatter plot relative increase in γH2AX following 60 min of recovery ofthe NHBE cells in particular phases of the cell cycle for mock exposed(upper plot) and for IM16 smoke exposed (lower plot) cells.

FIG. 19. Plots showing the increase (Δ) in mean γH2AX immunofluorescenceduring different time points of the recovery of A549 cells (per unit ofDNA) after exposure to smoke of IM16, Quest 3®, and Omni® cigarettes for20 minutes, calculated for cells in particular phases of the cell cycle.

FIG. 20. Bar plots showing the increase (Δ) in mean γH2AXimmunofluorescence of A549 cells (top) and NHBE cells (bottom) exposedto smoke of IM16 cigarettes for 20 minutes, followed by a 1 hourrecovery, for cells treated with phosphate-buffered saline (PBS) orN-acetyl-L-cysteine (NAC) during exposure (first value) and duringrecovery (second value).

FIG. 21. Bar plot showing the increase (Δ) in mean γH2AXimmunofluorescence of A549 cells exposed to smoke from IM16, Omni® andQuest 3® in the presence of PBS or NAC.

FIG. 22. Plot of the relative amount of mean γH2AX immunofluorescence ofA549 cells exposed to smoke from IM16 as a function of differentconcentrations of NAC, calculated for cells in particular phases of thecell cycle. Horizontal dashed line indicates 50% reduction in γH2AXimmunofluorescence. Vertical dashed lines indicate the estimated NACconcentration for each cell type at 50% reduction.

FIG. 23. Bar plots showing the increase (Δ) in mean γH2AXimmunofluorescence of A549 cells (upper plot) and NHBE cells (lowerplot) exposed to the vapor phase of smoke from IM16, Quest 1® and Quest3®, and smoke from IM16 in the presence of PBS or NAC.

FIG. 24. Bar plots showing the increase (Δ) in mean γH2AXimmunofluorescence of G₁, S and G₂M phase A549 cells (left plots) andG₁, S and G₂M phase NHBE cells (right plots) exposed to the vapor phaseof smoke from IM16, Quest 1® and Quest 3® in the presence of PBS or NAC.

FIG. 25. Bar plot showing the relative percent cloning efficiency ofA549 cells 5 days after exposure to smoke from IM16 or Marlboro® for 10,15 or 20 minutes.

FIG. 26. Bar plots showing the relative percent cloning efficiency ofA549 cells 5 days after exposure to smoke from IM16, Quest 1® or Quest3® for 10, 20 or 30 minutes (top two plots), or 6 days after (bottomplot) exposure to smoke from IM16, Marlboro® or Omni®, for 10, 15 or 20minutes.

FIG. 27. Bar plot showing the relative percent cloning efficiency ofA549 cells 5 days after exposure to smoke from IM16 for 20 minutes inthe presence of PBS or 1 mM, 5 mM, 10 mM or 25 mM NAC.

FIG. 28. Bar plot showing the relative percent cloning efficiency ofA549 cells 5 days after exposure to smoke from IM16, Omni® or Quest 3®for 20 minutes in the presence of PBS or 25 mM NAC.

FIG. 29. Bar plot showing the relative percent cloning efficiency ofA549 cells 5 days after exposure to vapor phase of smoke from IM16,Quest 1® or Quest 3®, or smoke of IM16 for 20 minutes in the presence ofPBS or 25 mM NAC.

FIG. 30. Bar plot of results from Example 2 showing the increase (A) inmean γH2AX immunofluorescence of A549 cells exposed to smoke from IM16,Omni® and Quest 3® in the presence of PBS or NAC, calculated for cellsin particular phases of the cell cycle.

FIG. 31. Plot depicting γH2AX associated fluorescence (γH2AX; X-axis)and the number of cells having the corresponding γH2AX fluorescencelevel (Y axis), for buccal cells of a subject subsequent to smoking acigarette (smoker) or a subject who did not smoke a cigarette(non-smoker).

FIG. 32. Bar plot of results from Example 2 showing the increase (Δ) inmean γH2AX immunofluorescence of A549 cells exposed to smoke from IM16,Marlboro®, Marlboro Light®, and Quest 3®, calculated for cells inparticular phases of the cell cycle.

FIG. 33. Bar plot of results from FIG. 32 showing the increase (Δ) inmean γH2AX immunofluorescence of A549 cells exposed to smoke from IM16,Marlboro®, Marlboro Light®, and Quest 3®, averaged for all cell cycles.

FIG. 34A is a Venn diagram comparing gene expression modulations inducedby cigarette smoke condensates of two different tobacco products (e.g.,cigarettes) CSC-A (3665) and CSC-B (3668). The number of genes uniquelyaffected by exposure to each product CSC-A (1226) and CSC-B (1229) isgiven in each sector. The intersections between sectors reflect thenumber of genes that are affected by both CSCs (2439).

FIG. 34B is a Venn diagram comparing gene expression modulations inducedby CSC-A (3665), CSC-B (3668), and S9 metabolic fraction (1680). Thenumber of unique genes affected by each treatment is given, CSC-A (992),CSC-B (1039), and S9 (383) and the intersections between sectors reflectthe number of genes that are affected by more than one treatment (e.g.,a common set of 873 genes is affected by CSC-A, CSC-B and S9).

FIG. 35A-C illustrate gene expression profiles between 0 and 12 hours,which are expressed a percent of highest expression value for each gene.F-cluster numbers are given at the top of each cluster of profiles. Thenumber of member genes in each cluster (n) is shown for each cluster.FIG. 35A shows Clusters that contain 50 or more genes in CSC-A-treatedcells. FIG. 35B shows Clusters containing 50 or more genes inCSC-B-treated cells. FIG. 35C shows Clusters containing 50 or more genesin S9-treated cells.

FIG. 36 illustrates a cluster analysis of genes that were hypervariable(HV) in all three treatment groups (A: CSC-A, B: CSC-B, and S9) in theform of a Dendrogram that depicts the hierarchical relationship betweenthe three treatments based on their gene expression patterns at all timepoints from 0-12 hours.

FIG. 37 shows correlation mosaics of the genes listed in Table 2.Correlation coefficients were generated for each of the 40 genes inTable 2, comparing the set to itself in each of the three conditions.The same gene order runs across the x and y axes of the mosaics.Correlation mosaics for HV genes highly correlated in response to CSC-Aand CSC-B, and not correlated with responses to S9. Each pixel in theplot represents a correlation coefficient of gene expression. Geneshighly positively correlated are denoted in gray and those highlynegatively correlated are in black. The same order of the genes alongaxis is used for all three mosaics. Genes highly correlated in CSC-A andCSC-B, but not in S9-treated cells are denoted as a gray cluster in thelower left hand corner of CSC-A and the CSC-B mosaic. This cluster isdisrupted in the S9 mosaic demonstrating the variance in gene regulationthat occurred in S9-treated cells.

FIG. 38 shows the functional associations of HV genes specific for CSC-Aand CSC-B treatment. The expression patterns of this set of genes arehighly correlated in CSC-treated NHBE cells and not correlated withthose seen in cells treated with S9 alone. Cross-hatched ovals indicategenes from Table 2 (i.e., HV genes specific for CSC-A and CSC-Btreatment). Ovals with slanted lines (indicating additional proteins notin Table 2) were added to better define the regulatory networks of thegenes identified in this analysis. Ovals with dashed lines indicateclasses of functional peptides. Rectangles indicate cellular processesin which these genes participate. Each line indicates a regulatoryrelationship (binding, regulation, etc.) based upon a literaturereference. Regulatory relationships are denoted in a box on the linewith positive regulation represented as a plus sign, negative regulationas a minus sign, and unknown relationships by no sign.

FIG. 39 shows the functional associations of genes, which are highlycorrelated in all three treatment groups (CSC-A, CSC-B, and S9). Thegenes, pathways, and functional interconnections among these elementsfor genes correlated in all three treatment groups are represented. Geneand pathway symbols are described in FIG. 38. Cross-hatched ovalsindicate genes from Table 3 (i.e., genes specific for S9 treatment).Ovals with slanted lines (indicate additional proteins not in Table 3),cross-hatched oval (cell object—DNA) and white triangle (indicatingsmall molecule—estrogen) were added to better define the regulatorynetworks of the genes identified in this analysis. Ovals with dashedlines indicate classes of functional peptides. White rectangles indicatecellular processes in which these genes participate. Each line indicatesa regulatory relationship (binding, regulation, etc.) based upon aliterature reference. Regulatory relationships are denoted in a box onthe line with positive regulation represented as a plus sign, negativeregulation as a minus sign, and unknown relationships by no sign.

FIG. 40 shows the results of a discriminant function analysis (DFA),which identified genes having high discriminatory capabilities. Valuesof the roots obtained by DFA analysis were used to graphically depictthe differences of the gene expression values obtained for the threetreatments (CSC-A, CSC-B, and S9). Root values for the 2-12 h timepoints for each treatment are represented by filled circles (CSC-A),open circles (CSC-B), and filled triangles (S9).

FIG. 41 shows the functional associations of genes, which are providedin Table 3. The genes, pathways, and functional interconnections amongthese elements for genes having the highest discriminatory potentialamong all three treatment groups are represented. Gene and pathwaysymbols are described in previous figures.

FIGS. 42A and B show a comparison of expression behavior of heat shockprotein family members DNAJA1 and DNAJB1 in Experiment 1 (FIG. 42A) and2 (FIG. 42B). Each time point represents the average of 2 or 3replicates per condition.

FIG. 43 is a hierarchical clustering of samples using 105 genes thatwere both over-expressed upon treatment of NHBE cells with CS in twoseparate experiments, and encoded protein products that modulate one ofthe 4 major CS-affected GO-defined cellular functions identified.Samples a-b are from Experiment 1, samples c-e are from Experiment 2. Abar indicates heat shock and heat shock-associated genes showing greatlyincreased expression exclusively at 4 h. Markings indicate genes whoseexpression is known to be regulated by transcription factor NRF2.

FIG. 44 shows a plot of γH2AX immunofluorescence in A549 cells exposedto smoke of different combinations of tobaccos and filters from IM16,Omni® and Quest 3® cigarettes, corrected according to the γH2AXimmunofluorescence for mock-exposed cells. FIG. 44A depicts γH2AXimmunofluorescence for the unmodified cigarettes. FIG. 44B depicts γH2AXimmunofluorescence for cigarettes containing IM16 tobacco and IM16,Omni® and Quest 3® filters. FIG. 44C depicts γH2AX immunofluorescencefor cigarettes containing Omni® tobacco and either an IM16 or Omni®filter. FIG. 44D depicts γH2AX immunofluorescence for cigarettescontaining Quest 3® tobacco and either an IM16 or Quest 3® filter.

FIG. 45 shows bar plots showing the relative percent cloning efficiencyof A549 cells 5 days after exposure to smoke of different combinationsof tobaccos and filters from IM16, Omni® or Quest 3® cigarettes,relative to mock cloning efficiency.

FIG. 46 shows bar plots showing the relative percent cloning efficiencyof A549 cells 5 days after exposure to smoke of different combinationsof tobaccos and filters from IM16, Quest 1® or Quest 3®, relative tomock cloning efficiency.

FIG. 47 shows bar plots showing the relative percent cloning efficiencyof A549 cells 5 days after exposure to smoke of different combinationsof tobaccos and filters from IM16, Omni® or Quest 3® cigarettes,relative to mock cloning efficiency.

DETAILED DESCRIPTION

I. Introduction

The health consequences of tobacco consumption are known but many peoplecontinue to use tobacco products. The addictive properties of tobaccoproducts are largely attributable to the presence of nicotine. Inaddition to being one of the most addictive substances known, nicotineis also a precursor for a large number of carcinogenic compounds presentin tobacco and the body. Many other harmful compounds in addition tonicotine are present in conventional tobacco, however.

There is currently a great interest in developing approaches to decreasethe levels of noxious, carcinogenic, or addictive substances includingtar, TSNAs, and nicotine in tobacco. Although researchers have developedseveral approaches to reduce some of these harmful compounds, manyconventional techniques result in a product that has poor taste,fragrance, or smoking properties. Some processes, for example, reducethe nicotine content of tobacco by microbial enzymatic degradation,chemical extraction, or high pressure extraction. (See e.g., U.S. Pat.Nos. 4,557,280; 4,561,452; 4,848,373; 4,183,364; and 4,215,706, all ofwhich are hereby expressly incorporated by reference in theirentireties). More recently, techniques in genetic engineering andchemically-induced gene suppression have been employed to make reducednicotine and/or reduced tobacco specific nitrosamine (TSNA) tobacco.(See e.g., Conkling et al., WO98/56923; U.S. Pat. Nos. 6,586,661;6,423,520; and U.S. patent application Ser. Nos. 09/963,340; 10/356,076;09/941,042; 10/363,069; 10/729,121; 10/943,346; Timko et al., WO00/67558, which designated the United States and was published inEnglish, Nakatani et al., U.S. Pat. Nos. 5,684,241; 5,369,023;5,260,205; and Roberts et al. U.S. Pat. No. 6,700,040, all of which arehereby expressly incorporated by reference in their entireties). In viewof the foregoing, and notwithstanding the various efforts exemplified inthe above reports, there remains a need for tobacco that has a reducedpotential to contribute to a tobacco-related disease and methods ofproducing such tobacco.

Embodiments provided herein relate to tobacco and/or tobacco productshaving a reduced amount of a harmful compound, and methods ofdeveloping, screening and using such tobacco and tobacco products.Several approaches are provided to reduce the amount of one or moreharmful compounds in tobacco by, for example, modifying the expressionof a gene that is involved in the production of a harmful compound intobacco. Also provided are methods of determining whether the removal ofa harmful compound yields a tobacco and/or a tobacco product that has areduced potential to contribute to a tobacco-related disease. Alsoprovided are reduced-risk tobacco and tobacco products made inaccordance with the methods provided herein. Also provided are methodsof using the reduced-risk tobacco and tobacco products made inaccordance with the methods provided herein.

II. Modified Tobacco

Several approaches to create a reduced risk tobacco product having areduced amount of a harmful compound are described. At least some of thereduced risk tobacco products provided herein contain modified tobacco.As used herein, “modified tobacco” refers to a tobacco that has beensubjected to one or more genetic, chemical or processing steps that isdifferent than the conventional treatment or processing of traditional“wild-type” tobacco products. In one example, a tobacco product can begenetically modified, by, for example, administering to a tobacco planta nucleic acid molecule that modulates expression of one or more genesin the tobacco plant that produce a compound. Genetically modifiedtobacco and methods of preparing same are provided elsewhere herein. Inanother example, a tobacco product can be chemically modified, by, forexample, extracting or chemically altering one or more components oftobacco, according to methods known in the art as exemplified in U.S.Pat. Nos. 6,789,548, 4,557,280; 4,561,452; 4,848,373; 4,183,364;4,215,706; 4,257,430; 4,248,251; 4,235,251; 4,216,784; 4,177,822;4,055,191 (all of which are herein expressly incorporated by referencein their entireties) or by adding one or more compounds to a tobaccoplant prior to harvesting the tobacco, as known in the art andexemplified in U.S. Pat. Pub. No. 20050072047, herein expresslyincorporated by reference in its entirety. Additional modified tobaccoscontemplated herein include reconstituted tobacco, extracted tobacco,and expanded or puffed tobacco. In some embodiments, the tobacco ismodified to have a reduced amount of a compound that contributes to atobacco-related disease, including, but not limited to, a compoundassociated with a tobacco-related disease or a metabolite thereof (e.g.,tobacco sterols, nicotine, a TSNA, and a gene product that is involvedin the production of a compound associated with a tobacco-relateddisease or a metabolite thereof).

The modified tobacco described herein is suitable for conventionalgrowing and harvesting techniques (e.g. topping or no topping, baggingthe flowers or not bagging the flowers, cultivation in manure rich soilor without manure) and the harvested leaves and stems are suitable foruse in any traditional tobacco product including, but not limited to,pipe, cigar and cigarette tobacco and chewing tobacco in any formincluding leaf tobacco, shredded tobacco or cut tobacco. It is alsocontemplated that the modified tobacco (e.g., reduced nicotine/TSNAand/or sterol tobacco) described herein can be processed and blendedwith conventional tobacco so as to create a wide-range of tobaccoproducts with varying amounts of nicotine, TSNAs, and/or sterols.

In some embodiments, the modified tobacco has reduced levels ofnicotine, nornicotine, and/or sterols in tobacco. Alkaloids such asnicotine and nornicotine are precursors for a number of harmfulcompounds that contribute to tobacco-related disease (e.g., the tobaccospecific nitrosamines (TSNAs): N′-nitrosonornicotine (NNN),N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB),4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal(NNA)-4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL),4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and/or4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid (iso-NNAC) andacrolein). Sterols are precursors for a number of harmful compounds,which are generated by pyrolysis of tobacco, that also contribute totobacco-related disease (e.g., polycyclic aromatic hydrocarbons (PAHs),such as benz[a]pyrene (BAP), heterocyclic hydrocarbons, terpenes,paraffins, aromatic amines, and aldehydes). Because the presence ofthese harmful compounds in tobacco contributes to tobacco-relateddisease, a modified tobacco that comprises a reduced amount of any oneof these compounds, as compared to a reference tobacco (e.g., theindustry standard reference tobacco IM16 (Philip Morris® USA) or the lowtar reference cigarette 2R4F or the ultra low tar cigarette 1R5F, whichare Kentucky reference cigarettes that can be obtained from the Tobaccoand Health Institute at the University of Kentucky), a conventionaltobacco (e.g., a commercially available tobacco of the same class (e.g.,“full-flavor” or “light” or “ultra-light”)) or a non-transgenic tobacco(e.g., a tobacco of the same variety, such as Burley, VirginiaFlue-cured, or Oriental, or strain, such as LA Burley 21, K326, Tn90,Djebe1174, as the transgenic tobacco prior to genetic modification) hasa reduced potential to contribute to a tobacco-related disease. Tobaccoproducts comprising the modified tobacco can also be analyzed by variousapproaches to confirm that the tobacco is “reduced risk,” as compared toa parental strain or a reference tobacco using one or more of the assaysdescribed herein or otherwise known in the art. This “reduced risk”modified tobacco can then be processed, optionally, sterilized orotherwise made substantially-free of microbes, and said tobacco can beincorporated into tobacco products, preferably, cigarettes, optionally,by an aseptic approach so as to not introduce microbes (e.g., bacteria,mold, yeast, and fungi) into the products. Tobacco products comprisingthe modified tobacco can then be packaged, optionally, by an asepticapproach in air-tight or microbe-free packaging so as to not introducemicrobes into the products.

In this manner, the conversion of alkaloid to TSNA, which results frommicrobial growth on the tobacco when microbes are introduced duringprocessing, packaging, and storage, is significantly reduced. By usingthe embodied tobacco preparative methods, which may include severalaseptic processing, manufacturing, and packaging procedures, one canmaintain an amount of total TSNA (e.g., the collective content of NNN,NAT, NAB, and NNK) in or delivered by (e.g., as measured by FTC or ISOmethodologies) a commercially available tobacco product of less than orequal to 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4μg/g, or 0.5 μg/g) for a period of at least 1 week, 1 month, or 1-5years after packaging or incorporation of the tobacco into a tobaccoproduct (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years).

In some embodiments, a modified tobacco comprising a reduced amount ofalkaloid (e.g., a reduced amount of nicotine, nornicotine, and/or TSNAs)is contacted with an exogenous nicotine so as to raise the level ofnicotine in the contacted transgenic tobacco in a controlled fashion. Bythis approach, nicotine levels in transgenic tobacco that comprises areduced amount of endogenous nicotine (i.e., nicotine that is producedby the transgenic plant from which the transgenic tobacco is obtained)can be selectively raised to levels that are commensurate withconventional full-flavor cigarettes, light cigarettes, or ultra-lightcigarettes. (See e.g., WO 2005/018307, which designates the UnitedStates and was published in English, herein expressly incorporated byreference in its entirety). For example, modified tobacco comprising areduced amount of endogenous nicotine and/or TSNAs can be contacted withan amount of exogenous nicotine that is at least, equal to, or more than0.3 mg/g-20.0 mg/g (nicotine/gram of tobacco). That is, modified tobaccocomprising a reduced amount of endogenous nicotine and/or TSNAs can becontacted with an amount of exogenous nicotine that is or delivers(e.g., as measured by FTC or ISO methodologies) at least, equal to, ormore than 0.3 mg/g, 0.4 mg/g, 0.5 mg/g, 0.6 mg/g, 0.7 mg/g, 0.8 mg/g,0.9 mg/g, 1.0 mg/g, 1.1 mg/g, 1.2 mg/g, 1.3 mg/g, 1.4 mg/g, 1.5 mg/g,1.6 mg/g, 1.7 mg/g, 1.8 mg/g, 1.9 mg/g, 2.0 mg/g, 2.1 mg/g, 2.2 mg/g,2.3 mg/g, 2.4 mg/g, 2.5 mg/g, 2.6 mg/g, 2.7 mg/g, 2.8 mg/g, 2.9 mg/g,3.0 mg/g, 3.1 mg/g, 3.2 mg/g, 3.3 mg/g, 3.4 mg/g, 3.5 mg/g, 3.6 mg/g,3.7 mg/g, 3.8 mg/g, 3.9 mg/g, 4.0 mg/g, 4.1 mg/g, 4.2 mg/g, 4.3 mg/g,4.4 mg/g, 4.5 mg/g, 4.6 mg/g, 4.7 mg/g, 4.8 mg/g, 4.9 mg/g, 5.0 mg/g,5.1 mg/g, 5.2 mg/g, 5.3 mg/g, 5.4 mg/g, 5.5 mg/g, 5.6 mg/g, 5.7 mg/g,5.8 mg/g, 5.9 mg/g, 6.0 mg/g, 6.1 mg/g, 6.2 mg/g, 6.3 mg/g, 6.4 mg/g,6.5 mg/g, 6.6 mg/g, 6.7 mg/g, 6.8 mg/g, 6.9 mg/g, 7.0 mg/g, 7.1 mg/g,7.2 mg/g, 7.3 mg/g, 7.4 mg/g, 7.5 mg/g, 7.6 mg/g, 7.7 mg/g, 7.8 mg/g,7.9 mg/g, 8.0 mg/g, 8.1 mg/g, 8.2 mg/g, 8.3 mg/g, 8.4 mg/g, 8.5 mg/g,8.6 mg/g, 8.7 mg/g, 8.8 mg/g, 8.9 mg/g, 9.0 mg/g, 9.1 mg/g, 9.2 mg/g,9.3 mg/g, 9.4 mg/g, 9.5 mg/g, 9.6 mg/g, 9.7 mg/g, 9.8 mg/g, 9.9 mg/g,10.0 mg/g, 10.1 mg/g, 10.2 mg/g, 10.3 mg/g, 10.4 mg/g, 10.5 mg/g, 10.6mg/g, 10.7 mg/g, 10.8 mg/g, 10.9 mg/g, 11.0 mg/g, 11.1 mg/g, 11.2 mg/g,11.3 mg/g, 11.4 mg/g, 11.5 mg/g, 11.6 mg/g, 11.7 mg/g, 11.8 mg/g, 11.9mg/g, 12.0 mg/g, 12.1 mg/g, 12.2 mg/g, 12.3 mg/g, 12.4 mg/g, 12.5 mg/g,12.6 mg/g, 12.7 mg/g, 12.8 mg/g, 12.9 mg/g, 13.0 mg/g, 13.1 mg/g, 13.2mg/g, 13.3 mg/g, 13.4 mg/g, 13.5 mg/g, 13.6 mg/g, 13.7 mg/g, 13.8 mg/g,13.9 mg/g, 14.0 mg/g, 14.1 mg/g, 14.2 mg/g, 14.3 mg/g, 14.4 mg/g, 14.5mg/g, 14.6 mg/g, 14.7 mg/g, 14.8 mg/g, 14.9 mg/g, 15.0 mg/g, 15.1 mg/g,15.2 mg/g, 15.3 mg/g, 15.4 mg/g, 15.5 mg/g, 15.6 mg/g, 15.7 mg/g, 15.8mg/g, 15.9 mg/g, 16.0 mg/g, 16.1 mg/g, 16.2 mg/g, 16.3 mg/g, 16.4 mg/g,16.5 mg/g, 16.6 mg/g, 16.7 mg/g, 16.8 mg/g, 16.9 mg/g, 17.0 mg/g, 17.1mg/g, 17.2 mg/g, 17.3 mg/g, 17.4 mg/g, 17.5 mg/g, 17.6 mg/g, 17.7 mg/g,17.8 mg/g, 17.9 mg/g, 18.0 mg/g, 18.1 mg/g, 18.2 mg/g, 18.3 mg/g, 18.4mg/g, 18.5 mg/g, 18.6 mg/g, 18.7 mg/g, 18.8 mg/g, 18.9 mg/g, 19.0 mg/g,19.1 mg/g, 19.2 mg/g, 19.3 mg/g, 19.4 mg/g, 19.5 mg/g, 19.6 mg/g, 19.7mg/g, 19.8 mg/g, 19.9 mg/g, and 20.0 mg/g (nicotine/gram tobacco). Insome of the aforementioned embodiments, the modified tobacco contactedwith the exogenous nicotine is a transgenic tobacco comprising, forexample, one or more of the isolated nucleic acids, isolated nucleicacid cassettes, or isolated nucleic acid constructs described herein.

Nicotine-containing fractions, nicotine, or nicotine salts of organicacids are added to the reduced-nicotine transgenic tobacco by contactingsaid tobacco (e.g., spraying or additive application), with or withoutpropylene glycol, solvent, flavoring, or water at any stage of theharvesting, curing, fermenting, aging, reconstituting, expanding, orotherwise processing of the tobacco, preferably at a stage that ispost-cure, when flavorings and additives are provided. By “exogenousnicotine” is meant nicotine, nicotine derivatives, nicotine analogs,nicotine-containing fractions (e.g., extracts of Nicotiana), andnicotine salts of organic acids obtained from a source outside of thetransgenic tobacco to which the exogenous nicotine is applied. In thismanner, a modified tobacco that provides virtually any amount ofnicotine can be obtained.

In some embodiments, the exogenous nicotine (e.g., commerciallyavailable nicotine salts, liquid, or a nicotine-containing extractprepared from a Nicotiana plant or portion thereof) is contacted with areduced-alkaloid modified tobacco (e.g., a transgenic tobacco comprisinga reduced amount of nicotine and/or TSNA as prepared as describedherein) after the modified tobacco has been made substantially free ofmicrobes (e.g., bacteria, yeast, mold, or fungi). The reduced alkaloidmodified tobacco can be made substantially-free of microbes (e.g., anaseptic preparation) by employing sterilization, heat treatment,pasteurization, steam treatment, gas treatment, and radiation (e.g.,gamma, microwave, and ultraviolet). The term “substantially-free ofmicrobes” in some contexts can mean an amount of bacteria, mold, fungi,or yeast that is reduced to the point that the conversion of nicotine ortotal alkaloid to TSNA is negligible (e.g., the resultant concentrationof or the amount of delivered or provided total TSNA (e.g., NNN, NNK,NAT, and NAB) in or delivered by a tobacco or tobacco product is equalto or below 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg/g, 0.2 μg/g, 0.3 μg/g, 0.4μg/g, or 0.5 μg/g) after prolonged storage (e.g., at least 1-30 days,30-90 days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5years, 1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0years-4 years, and 4.0 years-5.0 years)). The term “substantially-freeof microbes” also includes the term “substantially-free of bacteria,”which means in some contexts that the tobacco or tobacco product issubstantially-free of Arthrobacter, Proteus, nicotine oxidizingbacteria, such as P-34, Pseudomonas, Xantomonas, or Zoogloea strains ofbacteria. For example, a tobacco or tobacco product issubstantially-free of bacteria or a particular strain of bacteria whensaid tobacco or tobacco product has less than or equal to 20% of thebacteria or a specific strain of bacteria normally present on thetobacco or tobacco product in the absence of application of a techniqueto rid the tobacco or tobacco product of bacteria (e.g., less than orequal to 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, or 20%). With respect to modified tobaccodescribed herein, the term “substantially-free of bacteria” can refer totobacco or a tobacco product containing the modified tobacco that hasless than or equal to 20% of the bacteria normally present on the strainof tobacco prior to modification and/or application of a technique torid the tobacco or tobacco product of bacteria (e.g., less than or equalto 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, or 20%).

Once the exogenous nicotine has been contacted with the microbe-freemodified tobacco, it is preferably processed and packaged asepticallyand the tobacco product is maintained in an airtight container so as tonot re-introduce microbes that convert the exogenous nicotine to TSNAs.By using the aseptic processing, manufacturing, and packagingprocedures, described herein, one can maintain an amount of total TSNA(e.g., the collective content of NNN, NAT, NAB, and NNK) in acommercially available tobacco product or delivered by a commerciallyavailable tobacco product, which comprises exogenous nicotine, of lessthan or equal to 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g,0.4 μg/g, or 0.5 μg/g) for at least 1 week, 1 month, or 1-5 years afterpackaging (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years).In some embodiments, the exogenous nicotine is contacted with a modifiedtobacco and a collective content of NNN, NAT, NAB, and NNN that ispresent or delivered by the tobacco is less than or equal to 0.5 μg/g(e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g). Insome embodiments, a collective content of NNN, NAT, NAB, and NNN of lessthan or equal to 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g,0.4 μg/g, or 0.5 μg/g) in or delivered by a tobacco product containingsaid transgenic tobacco can be maintained for at least at least 1 week,1 month, or 1-5 years after packaging (e.g., at least 1-30 days, 30-90days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years,1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4years, and 4.0 years-5.0 years). An exemplary modified tobacco istransgenic tobacco comprising, for example, one of the nucleic acidconstructs described herein. Accordingly, several embodiments addressthe problem of gradually increasing TSNA levels in alkaloid-containingtobacco products by employing processing, storage, and packaging methodsthat reduce the amount of microbial flora on the tobacco, limit there-introduction of microbes during processing and maintain a reducedamount of microbes (e.g., bacteria) once the product is packaged,stored, and sold. Tobacco and tobacco products comprising modifiedtobacco having a reduced amount of endogenous nicotine and an amount ofexogenous nicotine can be analyzed by various methods to confirm thatsaid tobacco and said tobacco products are “reduced risk” or have lessof a potential to contribute to a tobacco-related disease, as comparedto the parent strain of tobacco having conventional amounts ofendogenous nicotine or a reference tobacco.

Tobacco products that comprise a modified tobacco described hereininclude “full-flavor,” “lights,” and “ultra light” cigarettes typicallyhaving both reduced levels of alkaloids and levels of alkaloidscommensurate with a level of alkaloid common to the particular class ofcigarette (i.e., a conventional amount of nicotine). The term “tobaccoproducts” includes, but is not limited to, smoking materials (e.g.,cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum, andlozenges.

The term “reduced risk tobacco product” or “reduced risk tobacco”includes, but is not limited to, a tobacco product or tobacco comprisinga modified tobacco that has a reduced amount of a compound thatcontributes to a tobacco-related disease, or increased amounts of acompound that reduces the harmful effects of a compound that contributesto a tobacco-related disease such as nicotine, nornicotine, a sterol, orthe metabolites thereof including, but not limited to, a TSNA, anacrolein, an aldehyde, or harmful compounds generated upon pyrolysis oftobacco, including but not limited to, PAH, BAP, a heterocyclichydrocarbon, or an aromatic amine, as compared to the amount of thesecompounds in or generated by a reference tobacco or reference tobaccoproduct (e.g., IM16, 2R4F or 1R5F), a commercially available tobaccoproduct of the same class (e.g., full-flavor, lights, and ultra-lights),or, preferably, a tobacco of the same variety (e.g., Burley, VirginiaFlue-cured, or Oriental) or strain (e.g., LA Burley 21, K326, Tn90,Djebe1174) as the transgenic tobacco prior to genetic modification). Forexample, a reduced risk tobacco or a reduced risk tobacco product caninclude a transgenic tobacco or a tobacco product comprising transgenictobacco that up-regulates fewer genes associated with a tobacco-relateddisease as compared to a reference tobacco or reference tobacco product(e.g., IM16, 2R4F or 1R5F), a commercially available tobacco product ofthe same class (e.g., full-flavor, lights, and ultra-lights), or,preferably, a tobacco of the same variety (e.g., Burley, VirginiaFlue-cured, or Oriental) or strain (e.g., LA Burley 21, K326, Tn90,Djebe1174) as the transgenic tobacco prior to genetic modification).

Nitrosamines and Tobacco-Specific Nitrosamines

The term nitrosamine generally refers to any of a class of organiccompounds with the general formula R₂NNO or RNHNO (where R denotes anamine-containing group). Nitrosamines are present in numerous foods andhave been found to be carcinogenic in laboratory animals. Thesecompounds are formed by nitrosation reactions of amines such as aminoacids and alkaloids with nitrites and/or nitrous oxides. By themselves,nitrosamines are not carcinogenic substances, but in mammalsnitrosamines undergo decomposition by enzymatic activation to formalkylating metabolites which appear to react with biopolymers toinitiate their tumorgenic effect. Thus, by reducing the amount ofnitrosamine intake, one has effectively reduced the carcinogenicpotential in humans.

Nitrosamines have been identified in tobacco, tobacco products, andtobacco smoke by the use of techniques such as gaschromatography-thermal energy analysis (GC-TEA). Some of thesenitrosamines have been identified as tobacco-specific nitrosamines(TSNAs). TSNAs are primarily formed by reactions between the two mostabundant alkaloids, nicotine and nornicotine, with nitrous oxides (NOx),and they account proportionately for the highest concentration ofnitrosamines in both tobacco products and in mainstream smoke. Of theTSNAs identified, and the subset that have been found to be present incigarette smoke, the most characterized is N-nitrosamine,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (N-nitrosamine-ketone),or NNK. When injected at relatively high doses, NNK is carcinogenic inrodents. Minimal amounts of TSNAs are found in green tobacco, indicatingthat TSNA formation may occur during processing steps such as curing,drying, fermentation, burning or storage of tobacco.

TSNA formation is attributed to chemical, enzymatic and bacterialinfluences during tobacco processing, particularly during curing,fermentation and aging. Nitrosation of nornicotine, anatabine, andanabasine gives the corresponding nitrosamines: N′-nitrosonornicotine(NNN), N′-nitrosoanatabine (NAT) and N′-nitrosoanabasine (NAB).Nitrosation of nicotine in aqueous solution affords a mixture of4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), NNN, and4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal (NNA). Less commonlyencountered TSNAs include NNAL(4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol), iso-NNAL(4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol, 11) and iso-NNAC(4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid, 12). See, U.S.Pat. No. 6,135,121, the entire disclosure of which is hereby expresslyincorporated by reference in its entirety.

TSNA levels are particularly high in chewing tobaccos and snuff. Thepartially anaerobic processes that occur during fermentation promote theformation of TSNAs from tobacco alkaloids by promoting increased nitritelevels; in particular, over-fermentation can increase TSNA levels insnuff by its effects on nitrate levels and microbial enzymatic activity.The reduction of the TSNA level in snuff in recent years has beenachieved by maintaining a better control over the bacterial content inthese products.

Since the nitrate level of tobacco is important for TSNA formation incigarette smoke, a significant reduction of TSNAs in smoke can beachieved by low-nitrate leaf and stem blends. However, these methods maynegatively impact the smokability or the taste of the tobacco. The TSNAcontent of mainstream smoke can be reduced by as much as 80% bycellulose acetate filters, and it can be reduced still further by filterventilation.

Air-cured tobaccos such as Burley and dark-fired may have higher levelsof TSNAs than certain types of Flue-cured bright, Burley, or darktobaccos apparently because the high temperatures associated withflue-curing can kill the micro-organisms that transform the alkaloidsinto TSNAs. In air-cured types, nitrate (N—NO₃) is more abundant in theleaf (particularly in the leaf and stems) than in Flue-cured tobacco andthe alkaloid content is also much higher. This N—NO₃ is reduced tonitrite (NO₂ ⁻) by microbes during curing and the NO₂ ⁻ can be furtherreduced to NOx or react directly with alkaloids to form TSNAs.

It is contemplated that, in addition to the techniques described above,nitrate levels in tobacco (especially in the leaf) can be reduced bylimiting exposure to nitrosating agents or conditions. Air-curingexperiments at a higher temperature have shown that considerably higherlevels of N-nitrosamines are formed at a curing temperature of 32° C.than at 16° C., which is associated with a rise of the nitrite level inthe tobacco, and may also be associated with a rise in microbialenzymatic activity. Modified curing that involves faster drying fromwider spacing or from more open curing structures has been shown toreduce TSNA levels in Burley tobacco. The climatic conditions prevailingduring curing exert a major influence on N-nitrosamine formation, andthe relative humidity during air-curing can be of importance. Stalkcuring results in higher TSNA levels in the smoke than primed-leafcuring. Sun-cured Oriental tobaccos have lower TSNA levels than flue-and air-cured dark tobaccos. Accelerated curing of crude tobaccos suchas homogenized leaf curing limits the ability of bacteria to carry outthe nitrosation reactions. However, many of the methods described abovefor reducing TSNAs in Burley tobacco can have undesirable effects ontobacco taste.

TSNA formation in Flue-cured tobacco also results from exposure of thetobacco to combustion gases during curing, where nearly all of the TSNAsin Flue-cured tobacco (e.g., Virginia Flue-cured) result from a reactioninvolving NOx and nicotine. The predominant source of NOx is the mixtureof combustion gases in direct-fired barns. At present, Flue-curedtobacco is predominantly cured in commercial bulk barns. As a result ofenergy pressures in the U.S. during the 1960's, farmer-built “stickbarns” with heat-exchanged flue systems were gradually replaced withmore energy efficient bulk barns using direct-fired liquid propane gas(LPG) burners. These LPG direct-fired burner systems exhaust combustiongases and combustion by-products directly into the barn where contact ismade with the curing tobacco. Studies indicate that LPG combustionby-products react with naturally occurring tobacco alkaloids to formTSNA.

In contrast to direct-fired curing, heat-exchange burner configurationscompletely vent combustion gases and combustion by-products to theexternal atmosphere rather than into the barn. The heat-exchange processprecludes exposure of the tobacco to LPG combustion by-products, therebyeliminating an important source of nitrosating agent for TSNA formation,without degrading leaf quality or smoking quality. The use of heatexchangers reduces TSNA levels by about 90%. Steps are being taken toreduce TSNA levels in US tobacco by converting barns to indirect heatthrough the use of a heat exchanger, but these methods are veryexpensive. Although many of the approaches described in this sectionhave significant drawbacks, it should be understood that any or all ofthese techniques can be used with other techniques, as described herein,to make tobacco and tobacco products having reduced TSNAs. The sectionbelow provides more detail on nicotine and approaches to reduce nicotinein tobacco.

Nicotine

Nicotine is formed primarily in the roots of the tobacco plant and issubsequently transported to the leaves, where it is stored (Tso,Physiology and Biochemistry of Tobacco Plants, pp. 233-34, Dowden,Hutchinson & Ross, Stroudsburg, Pa. (1972)). Classical crop breedingtechniques have produced tobacco with lower levels of nicotine,including varieties with as low as 8% of the amount of nicotine found inwild-type tobacco. The many methods described herein can be used withvirtually any tobacco variety but are preferably used with Burley,Oriental or Flue-cured (e.g., Virginia Flue-cured) varieties.

Nicotine is produced in tobacco plants by the condensation of nicotinicacid and 4-methylaminobutanal. Two regulatory loci (Nic1 and Nic2) actas co-dominant regulators of nicotine production. Enzyme analyses ofroot tissue from single and double Nic mutants show that the activitiesof two enzymes, quinolate phosphoribosyl transferase (“QPTase”) andputrescene methyl transferase (PMTase), are directly proportional tolevels of nicotine biosynthesis. An obligatory step in nicotinebiosynthesis is the formation of nicotinic acid from quinolinic acid, astep that is catalyzed by QPTase. QPTase appears to be a rate-limitingenzyme in the pathway supplying nicotinic acid for nicotine synthesis intobacco. (See, eg., Feth et al., Planta, 168, pp. 402-07 (1986) andWagner et al., Physiol. Plant., 68, pp. 667-72 (1986), herein expresslyincorporated by reference in its entirety). A comparison of enzymeactivity in tobacco tissues (root and callus) with different capacitiesfor nicotine synthesis shows that QPTase activity is strictly correlatedwith nicotine content (Wagner and Wagner, Planta 165:532 (1985), hereinexpressly incorporated by reference in its entirety). In fact, Saundersand Bush (Plant Physiol 64:236 (1979), herein expressly incorporated byreference in its entirety), showed that the level of QPTase in the rootsof low nicotine mutants is proportional to the level of nicotine in theleaves.

The modification of nicotine levels in tobacco plants by antisenseregulation of putrescene methyl transferase expression has been proposedin U.S. Pat. Nos. 5,369,023 and 5,260,205, to Nakatani and Malik, and inPCT application WO 94/28142 and U.S. Pat. No. 5,668,295 to Wahad andMalik, which describe DNA encoding PMT and the use of sense andantisense PMT constructs, the entire disclosures of each of which arehereby expressly incorporated by reference in their entireties. Othergenetic modifications proposed to reduce nicotine levels are describedin PCT application WO 00/67558, to Timko, and WO 93/05646, to Davis andMarcum; the entire contents of each are hereby expressly incorporated byreference in their entireties. Although these investigators madesignificant contributions, there were significant drawbacks to theirexperimental design.

Provided herein are tobacco and tobacco products in which a plurality ofgenes involved in nicotine biosynthesis are inhibited. Most notably, itis presently revealed that there are several different PMT genes andeach may play a role in nicotine biosynthesis. Knocking-out only one PMTgene may create a leaky system allowing the other genes to compensatefor the reduction in nicotine biosynthesis. Accordingly, the PMTconstructs described herein were designed to inhibit a plurality ofdifferent PMT genes. That is, in some embodiments, the PMT constructsdescribed herein are designed to complement common regions to all fiveof the PMT genes so that inhibition of each of the PMT genes can beaccomplished with a single construct. Although many of the approachesdescribed in this section have significant drawbacks, it should beunderstood that any or all of these techniques can be used with othertechniques, as described herein, to make tobacco and tobacco productshaving reduced nicotine. The section below explains several approachesto reduce the amount of nicotine and sterols in tobacco and tobaccoproducts.

Reducing the Amount of Nicotine and Sterols in Tobacco

As discussed above, TSNAs, nicotine, nornicotine, and sterols contributesignificantly to tobacco-related disease, most notably the carcinogenicpotential of tobacco and tobacco products. Thus, tobacco and tobaccoproducts that have or produce reduced amounts of these compounds arereduced risk compositions (e.g., products that have a reduced potentialto contribute to a tobacco-related disease). Without wishing to be boundby any particular theory, it is contemplated that the creation oftobacco plants, tobacco and tobacco products that have a reduced amountof nicotine will also have reduced amounts of TSNAs. That is, byremoving nicotine from tobacco plants, tobacco and tobacco products, oneeffectively removes the most significant alkaloid substrate for TSNAformation. It was found that the reduction of nicotine in tobacco wasdirectly related to the reduction of TSNAs. Similarly, it iscontemplated that by removing sterols from tobacco, one can reduce theamount of PAHs generated from pyrolysis of the tobacco. Unexpectedly,the methods described herein not only produce tobacco with a reducedaddictive potential but, concomitantly, produce a tobacco that has areduced potential to contribute to a tobacco related disease.

It should be emphasized that the phrase “a reduced amount” as applied tonicotine and/or TSNAs is intended to refer to an amount of nicotineand/or TSNAs in a treated or transgenic tobacco plant, tobacco or atobacco product that is less than what would be found in a tobaccoplant, tobacco or a tobacco product from the same variety of tobacco,processed in the same manner, which has not been treated or was not madetransgenic for reduced nicotine and/or TSNAs. Thus, in some contexts,wild-type tobacco of the same variety that has been processed in thesame manner is used as a control by which to measure whether a reductionin nicotine, nornicotine, a sterol and/or TSNAs or PAHs has beenobtained by the inventive methods described herein.

The amount of TSNAs (e.g., collective content of NNN, NAT, NAB, and NNK)and nicotine in wild-type tobacco varies significantly depending on thevariety and the manner it is grown, harvested and cured. For example, acured Burley tobacco leaf can have approximately 30,000 parts permillion (ppm) nicotine and 8,000 parts per billion (ppb) TSNA (e.g.,collective content of NNN, NAT, NAB, and NNK); a Flue-cured leaf canhave approximately 20,000 ppm nicotine and 300 ppb TSNA (e.g.,collective content of NNN, NAT, NAB, and NNK); and an Oriental curedleaf can have approximately 10,000 ppm nicotine and 100 ppb TSNA (e.g.,collective content of NNN, NAT, NAB, and NNK). Tobacco having a reducedamount of nicotine and/or TSNA, can have no detectable nicotine and/orTSNA (e.g., collective content of NNN, NAT, NAB, and NNK), or maycontain some detectable amounts of one or more of the TSNAs and/ornicotine, so long as the amount of nicotine and/or TSNA is less thanthat found in tobacco of the same variety, grown under similarconditions, and cured and/or processed in the same manner. That is,cured Burley tobacco, as described herein, having a reduced amount ofnicotine can have between 0 and 30,000 ppm nicotine and 0 and 8,000 ppbTSNA, desirably between 0 and 20,000 ppm nicotine and 0 and 6,000 ppbTSNA, more desirably between 0 and 10,000 ppm nicotine and 0 and 5,000ppb TSNA, preferably between 0 and 5,000 ppm nicotine and 0 and 4,000ppb TSNA, more preferably between 0 and 2,500 ppm nicotine and 0 and2,000 ppb TSNA and most preferably between 0 and 1,000 ppm nicotine and0 and 1,000 ppb TSNA. Embodiments of cured Burley leaf prepared by themethods described herein can also have between 0 and 1000 ppm nicotineand 0 and 500 ppb TSNA, 0 and 500 ppm nicotine and 0 and 250 ppb TSNA, 0and 250 ppm nicotine and 0 and 100 ppb TSNA, 0 and 100 ppm nicotine and0 and 50 ppb TSNA, 0 and 50 ppm nicotine and 0 and 5 ppb TSNA and someembodiments of cured Burley leaf described herein have virtually nodetectable amount of nicotine or TSNA. In some embodiments above, theamount of TSNA refers to the collective content of NNN, NAT, NAB, andNNK.

Similarly, a Flue-cured tobacco embodiment having a reduced amount ofnicotine can have between 0 and 20,000 ppm nicotine and 0 and 300 ppbTSNA, desirably between 0 and 15,000 ppm nicotine and 0 and 250 ppbTSNA, more desirably between 0 and 10,000 ppm nicotine and 0 and 200 ppbTSNA, preferably between 0 and 5,000 ppm nicotine and 0 and 150 ppbTSNA, more preferably between 0 and 2,500 ppm nicotine and 0 and 100 ppbTSNA and most preferably between 0 and 1,000 ppm nicotine and 0 and 50ppb TSNA. Embodiments of Flue-cured tobacco, as described herein, canalso have between 0 and 500 ppm nicotine and 0 and 25 ppb TSNA, 0 and200 ppm nicotine and 0 and 10 ppb TSNA, 0 and 100 ppm nicotine and 0 and5 ppb TSNA and some embodiments of Flue-cured tobacco have virtually nodetectable amount of nicotine or TSNA. In some embodiments above, theamount of TSNA refers to the collective content of NNN, NAT, NAB, andNNK.

Further, a cured Oriental tobacco embodiment having a reduced amount ofnicotine can have between 0 and 10,000 ppm nicotine and 0 and 100 ppbTSNA, desirably between 0 and 7,000 ppm nicotine and 0 and 75 ppb TSNA,more desirably between 0 and 5,000 ppm nicotine and 0 and 50 ppb TSNA,preferably between 0 and 3,000 ppm nicotine and 0 and 25 ppb TSNA, morepreferably between 0 and 1,500 ppm nicotine and 0 and 10 ppb TSNA andmost preferably between 0 and 500 ppm nicotine and no detectable TSNA.Embodiments of cured Oriental tobacco can also have between 0 and 250ppm nicotine and no detectable TSNA and some embodiments of curedOriental tobacco have virtually no detectable amount of nicotine orTSNA. In some embodiments above, the amount of TSNA refers to thecollective content of NNN, NAT, NAB, and NNK.

Some embodiments comprise cured tobaccos (e.g., Burley, Flue-cured, orOriental) with reduced amounts of nicotine as compared to controlvarieties, wherein the amount of nicotine in or delivered by the product(e.g., as measured by FTC or ISO methodologies) is less than about 2mg/g, 1 mg/g, 0.75 mg/g, 0.5 mg/g or desirably less than about 0.1 mg/g,and preferably less than 0.08 mg/g, 0.07 mg/g, 0.06 mg/g, 0.05 mg/g,0.04 mg/g, 0.03 mg/g, 0.02 mg/g, 0.01 mg/g. Tobacco products made fromthese reduced nicotine and TSNA tobaccos are also embodiments. The term“tobacco products” include, but are not limited to, smoking materials(e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum,and lozenges. As mentioned above, these reduced nicotine and TSNAtobaccos can be treated with exogenous nicotine so as to incrementallyincrease the amount of nicotine in the product and by employing asepticprocessing and packaging techniques, the amounts of total TSNAs in theproduct can be kept at or below 0.5 μg/g for prolonged periods of time.

In some contexts, the phrase “reduced amount of nicotine and/or TSNAs”refers to the tobacco plants, cured tobacco, and tobacco products, asdescribed herein, which have less nicotine and/or TSNAs (e.g., thecollective content of NNN, NAT, NAB, and NNK) by weight than the samevariety of tobacco grown, processed, and cured in the same way. Forexample, wild type cured tobacco can have has approximately 1-4% dryweight nicotine and approximately 0.2%-0.8% dry weight TSNA depending onthe manner it was grown, harvested and cured. A typical cigarette hasbetween 2-11 mg of nicotine and approximately 5.0 μg of TSNAs. Thus, thetobacco plants, tobacco and tobacco products provided herein can have ordeliver, in dry weight for example, less than 0.01%, 0.015%, 0.02%,0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%,0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.175%, 0.2%,0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.35%, 0.375%, 0.4%, 0.425%, 0.45%,0.475%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%,and 1.0% nicotine and less than 0.01%, 0.015%, 0.02%, 0.025%, 0.03%,0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, and0.08% TSNA (e.g., collective content of NNN, NAT, NAB, and NNK).

Alternatively, a cigarette provided herein can have or deliver, forexample, less than 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg,0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg, 1.15 mg, 1.2 mg, 1.25 mg,1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg, 1.55 mg, 1.6 mg, 1.65 mg, 1.7mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg, 1.95 mg, 2.0 mg, 2.1 mg, 2.15 mg,2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg, 2.4 mg, 2.45 mg, 2.5 mg, 2.55 mg, 2.6mg, 2.65 mg, 2.7 mg, 2.75 mg, 2.8 mg, 2.85 mg, 2.9 mg, 2.95 mg, 3.0 mg,3.1 mg, 3.15 mg, 3.2 mg, 3.25 mg, 3.3 mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5mg, 3.55 mg, 3.6 mg, 3.65 mg, 3.7 mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg,3.95 mg, 4.0 mg, 4.1 mg, 4.15 mg, 4.2 mg, 4.25 mg, 4.3 mg, 4.35 mg, 4.4mg, 4.45 mg, 4.4 mg, 4.45 mg, 4.5 mg, 4.55 mg, 4.6 mg, 4.65 mg, 4.7 mg,4.75 mg, 4.8 mg, 4.85 mg, 4.9 mg, 4.95 mg, 5.0 mg, 5.5 mg, 5.7 mg, 6.0mg, 6.5 mgmg, 6.7 mg, 7.0 mg, 7.5 mg, 7.7 mg, 8.0 mg, 8.5 mg, 8.7 mg,9.0 mg, 9.5 mg, 9.7 mg, 10.0 mg, 10.5 mg, 10.7 mg, and 11.0 mg nicotineand less than 0.001 μg, 0.002 μg, 0.003 μg, 0.004 μg, 0.005 μg, 0.006μg, 0.007 μg, 0.008 μg, 0.009 μg, 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg,0.05 μg, 0.06 μg, 0.07 μg, 0.08 μg, 0.09 μg, 0.1 μg, 0.15 μg, 0.2 μg,0.25 μg, 0.3 μg, 0.336 μg, 0.339 μg, 0.345 μg, 0.35 μg, 0.375 μg, 0.4μg, 0.414 μg, 0.45 μg, 0.5 μg, 0.515 μg, 0.55 μg, 0.555 μg, 0.56 μg,0.578 μg, 0.58 μg, 0.6 μg, 0.611 μg, 0.624 μg, 0.65 μg, 0.7 μg, 0.75 μg,0.8 μg, 0.85 μg, 0.9 μg, 0.95 μg, 1.0 μg, 1.1 μg, 1.114 μg, 1.15 μg, 1.2μg, 1.25 μg, 1.3 μg, 1.35 μg, 1.4 μg, 1.45 μg, 1.5 μg, 1.55 μg, 1.6 μg,1.65 μg, 1.7 μg, 1.75 μg, 1.8 μg, 1.85 μg, 1.9 μg, 1.95 μg, 2.0 μg, 2.1μg, 2.15 μg, 2.2 μg TSNA (e.g., collective content of NNN, NAT, NAB, andNNK).

Unexpectedly, it was discovered that several methods for reducingendogenous levels of nicotine in a plant are suitable for producingtobacco that is substantially free of nitrosamines, especially TSNAs.Any method that reduces levels of other alkaloids, includingnorniticotine, is likewise suitable for producing tobacco substantiallyfree of nitrosamines, especially TSNAs. As described, embodimentscomprise methods of reducing the carcinogenic potential of a tobaccoproduct comprising providing a cured tobacco as described herein andpreparing a tobacco product from said cured tobacco, whereby thecarcinogenic potential of said tobacco product is thereby reduced.

In some embodiments that employed the A622 inhibition construct, it wasfound that transgenic tobacco that had conventional levels of nicotinebut significantly reduced levels of nornicotine were produced. Thisparticular line of tobacco is particularly useful because nornicotinemay be the most significant precursor for NNN in tobacco. Accordingly,reduced risk conventional cigarettes and other tobacco products (e.g.,snuff) comprising the A622 inhibition construct are embodiments.

Other embodiments include the use of the cured tobacco described hereinfor the preparation of a tobacco product that contains reduced amountsof carcinogens as compared to control varieties and/or that reduces theamount of a TSNA or TSNA metabolite in a human that uses tobacco. Insome embodiments, for example, the tobacco smoking products describedherein reduce the carcinogenic potential of side stream or main streamtobacco smoke in humans exposed to said side stream or main streamtobacco smoke. By providing the modified cured tobacco described hereinin a product that undergoes pyrolysis, for example, the side streamand/or main stream smoke produced by said product comprises a reducedamount of TSNAs and/or nicotine. Thus, the cured tobacco describedherein can be used to prepare a tobacco smoking product that comprises areduced amount of TSNAs in side stream and/or mainstream smoke.

In the United States, tar, nicotine, and carbon monoxide yields areobtained using the Federal Trade Commission (FTC) smoking-machine testmethod, which defines the measurement of tar as that material capturedby a Cambridge pad when a cigarette is machine smoked, minus nicotineand water (Pillsbury, et al., 1969, “Tar and nicotine in cigarettesmoke”. J. Assoc. Off. Analytical Chem., 52, 458-62). Specifically, theFTC cigarette-testing method collects smoke samples by simulatingpuffing volumes of 35 ml of cigarette smoke for two seconds every 58seconds, with none of the filter ventilation holes blocked (if any),until the burn line reaches the tipping paper plus 2 mm, or a line drawn23 mm from the end of a non-filter cigarette. This FTC smoking-machinetest method has been used in the United States since 1967 to determinesmoke cigarette yields for tar and nicotine. The determination of carbonmonoxide yields in cigarette smoke was added to this method in 1980.

In 1967, when the FTC introduced its testing method, it issued a newsrelease and explained that the purpose of the testing “is not todetermine the amount of tar and nicotine inhaled by any human smoker,but rather to determine the amount of tar and nicotine generated when acigarette is smoked by a machine in accordance with the prescribedmethod.” Nevertheless, the method serves an important role in providingan accurate way to rank and compare cigarettes according to tar,nicotine and carbon monoxide yields.

The International Standards Organization (ISO) developed a very similarsmoking-machine test method for tar, nicotine, and carbon monoxideyields of cigarettes (ISO, 1991 “Cigarettes—determination of total andnicotine-free dry particulate matter using a routine analytical smokingmachine” ISO: 4387:1991).

The FTC and ISO smoking methods differ in the following eight areas.

-   -   The FTC method specifies laboratory environmental conditions of        75° F.±1° F. (23.8° C.±1° C.) and a relative humidity of 60%±2%        for both the equilibration and testing. The time of        equilibration is a minimum of 24 hours and a maximum of 14 days.        This is compared to the ISO specifications of 22° C.±1° C. and        60%±2% relative humidity for equilibration, 22° C.±2° C. and 60%        relative humidity±5% for testing. The equilibration time is a        minimum of 48 hours and a maximum of 10 days.    -   The FTC defines the cigarette butt length as a minimum of 23        millimeters or the tipping paper plus three millimeters        whichever is longer. ISO defines butt length as the longest of        23 millimeters or tipping paper plus three millimeters or the        filter plus eight millimeters. Both methods specify a        23-millimeter butt length for non-filter cigarettes.    -   ISO defines the position of the ashtray at 20-60 millimeters        below the cigarettes in the smoking machine. FTC does not        specify a position.    -   ISO specifies a two-piece snap together reusable filter holder.        This filter holder contains the Cambridge pad and uses a        synthetic rubber perforated washer to partly obstruct the butt        end of the cigarette. The FTC method defines the use of a        Cambridge filter pad but does not specify a filter pad holder        assembly.    -   The ISO method specifies airflow across the cigarettes at the        cigarette level. FTC specifies the use of a monitor cigarette to        adjust airflow.    -   The ISO procedure defines the process of wiping the excess total        particulate matter (TPM) out of the used filter holder. The        inner surfaces of the filter holder are wiped with two separate        quarters of an unused conditioned filter pad. The FTC method        uses the backside (the side opposite of the trapped TPM) to wipe        the inner surface of the filter holder.    -   ISO specifies using 20 ml per Cambridge pad of extraction        solution to analyze nicotine and water in TPM. The FTC procedure        defines 10 ml per Cambridge pad.    -   ISO defines the internal standards for the gas chromatographic        determination of nicotine and water. The FTC procedure does not        specify the internal standards.

These differences typically result in slightly lower measured deliveriesfor the ISO Method versus the FTC Method. The measured values betweenFTC and ISO methods are within the detection limits of the test or aboutno greater than 0.4 mg tar and about 0.04 mg nicotine for cigarettesthat yield over about 10 mg.

In some embodiments, for example, the collective content of NNN, NAT,NAB, and NNK in the mainstream or side stream smoke from a tobaccoproduct comprising the modified tobacco, including genetically modifiedtobacco, described herein is between about 0-5.0 μg/g, 0-4.0 μg/g, 0-3.0μg/g, 0-2.0 μg/g, 0-1.5 μg/g, 0-1.0 μg/g, 0-0.75 μg/g, 0-0.5 μg/g,0-0.25 μg/g, 0-0.15 μg/g, 0-0.1 μg/g, 0-0.05 μg/g, 0-0.02 μg/g, 0-0.015μg/g, 0-0.01 μg/g, 0-0.005 μg/g, 0-0.002 μg/g, or 0-0.001 μg/g. That is,some embodiments are genetically modified Burley tobacco, wherein theside stream or mainstream smoke produced from a tobacco productcomprising said Burley tobacco has a collective content of NNN, NAT,NAB, and NNK in the mainstream or side stream smoke between about 0-5.0μg/g, 0-4.0 μg/g, 0-3.0 μg/g, 0-2.0 μg/g, 0-1.5 μg/g, 0-1.0 μg/g, 0-0.75μg/g, 0-0.5 μg/g, 0-0.25 μg/g, 0-0.15 μg/g, 0-0.1 μg/g, 0-0.05 μg/g,0-0.02 μg/g, 0-0.015 μg/g, 0-0.01 μg/g, 0-0.005 μg/g, 0-0.002 μg/g, or0-0.001 μg/g.

Other embodiments concern modified Flue-cured tobacco, such asgenetically modified Flue-cured tobacco, wherein the sidestream ormainstream smoke produced from a tobacco product comprising saidFlue-cured tobacco has a collective content of NNN, NAT, NAB, and NNK inthe mainstream or side stream smoke between about 0-5.0 μg/g, 0-4.0μg/g, 0-3.0 μg/g, 0-2.0 μg/g, 0-1.5 μg/g, 0-1.0 μg/g, 0-0.75 μg/g, 0-0.5μg/g, 0-0.25 μg/g, 0-0.15 μg/g, 0-0.1 μg/g, 0-0.05 μg/g, 0-0.02 μg/g,0-0.015 μg/g, 0-0.01 μg/g, 0-0.005 μg/g, 0-0.002 μg/g, or 0-0.001 μg/g.

More embodiments concern modified Oriental tobacco, wherein thesidestream or mainstream smoke produced from a tobacco productcomprising said Oriental tobacco has a collective content of NNN, NAT,NAB, and NNK in the mainstream or side stream smoke between about 0-5.0μg/g, 0-4.0 μg/g, 0-3.0 μg/g, 0-2.0 μg/g, 0-1.5 μg/g, 0-1.0 μg/g, 0-0.75μg/g, 0-0.5 μg/g, 0-0.25 μg/g, 0-0.15 μg/g, 0-0.1 μg/g, 0-0.05 μg/g,0-0.02 μg/g, 0-0.015 μg/g, 0-0.01 μg/g, 0-0.005 μg/g, 0-0.002 μg/g, or0-0.001 μg/g.

Additional Tobacco Modifications

Additional modified tobaccos that can be used in the methods and tobaccoproducts provided herein include, but are not limited to, chemicallymodified tobacco, expanded, extracted, or puffed tobacco, andreconstituted tobacco.

Any of a variety of chemically modified tobaccos can be included in themethods and tobacco products provided herein. For example, the chemicalmodification can include palladium, or can include an auxin, auxinanalog, or jasmonate antagonist (see e.g., U.S. Pat. No. 6,789,548 andU.S. Pat. App. Pub. No. 20050072047, both of which are hereby expresslyincorporated by reference in their entirety).

By one approach, a chemically modified tobacco is made as follows. Atobacco is provided and a casing solution is applied thereto.Thereafter, a plurality of metallic or carbonaceous catalytic particleshaving a mean average or a mode average particle size of less than about20 microns is applied to the tobacco in a form separate from the casingsolution. Next, a nitrate or nitrite source in a form separate from thecasing solution and in a form separate from the plurality of metallic orcarbonaceous catalytic particles is applied to the tobacco, before,after or simultaneously with applying the plurality of particles butafter applying the casing solution, whereby a smoking composition isobtained. In some embodiments of this modified tobacco, a polyaromatichydrocarbon, azaarene, carbazole, or a phenolic compound is reduced.Using this approach, the Omni® tobacco product was developed.

By another approach, a chemically modified tobacco is made byidentifying a tobacco plant in a field for nicotine reduction; andcontacting said tobacco plant with a composition selected from the groupconsisting of an auxin, auxin analog, and jasmonate antagonist frombetween about 21 days before topping to about 21 days after topping saidtobacco plant, whereby the amount of nicotine in said topped tobaccoplant contacted with said composition is below that of a topped tobaccoplant of the same variety, grown under the same conditions, which hasnot been contacted with said composition.

In another example, the chemically modified tobacco can be extractedtobacco. By some approaches the chemically modified tobacco is extractedwith an organic solvent and other processes use super-critical fluidextraction or carbon dioxide. In another example, the chemicalmodification can be a biotic modification. Microbes that ingest nitratesand alkaloids can be applied to tobacco so as to obtain a reducednicotine tobacco; for example such a biotic modification can includebacteria. In another example, the tobacco is processed to remove thepresence of a microbe. In another example the chemically modifiedtobacco can be sterilized, pasteurized, or radiated.

In another example, the chemically modified tobacco can have addedthereto an exogenous component of tobacco or analog thereof. Tobacco canbe modified to increase or decrease one or more compounds such asproteins, metabolites, nicotine-related compounds and sterols. In somemethods provided herein, a tobacco which has been modified to producelower levels of one or more compounds such as nicotine or a nicotinemetabolite, or a sterol, can have exogenously added thereto, one ofthese lower-level compounds, one or more but not all lower-levelcompounds, or all lower-level compounds or an analog of the compound(s).

Such tobaccos with one or more exogenously added compounds can becompared in accordance with the methods provided herein to the sametobacco to which no exogenous compound has been added, to which adifferent exogenous compound has been added, or to which a differentlevel of the same exogenous compound has been added. For example, themethods provided herein can be used to compare a tobacco that has beengenetically modified to produce reduced nicotine levels with the sametobacco to which exogenous nicotine or a nicotine analog has been addedthereto. By performing such methods, the role of the exogenously addedcompound on cell damage or other response determined according to themethods provided herein (e.g., apoptosis or cell proliferation), can bedetermined.

In another example, the chemically modified tobacco has had addedthereto a compound or composition containing antioxidants. Tobacco atany stage of its processing can have added thereto an antioxidantcompound or a composition with antioxidant properties. Any of a varietyof known antioxidant compounds can be added to the tobacco, including,but not limited to, lycopene, tocopherol, tocopherol metabolites,ascorbic acid, unsaturated fatty acids, N-acetyl cysteine, and otherantioxidants known in the art. A composition with antioxidant propertiescan include a biological composition or extract that can neutralizeoxidants, such as milk or milk proteins, tumeric or tumeric extracts,barley or barley extracts, alfalfa or alfalfa extracts. Other compoundsthat can be added to the tobacco include thiol-containing proteins,plant extracts, aromatic compounds (e.g., caffeine or pentoxyfyllen,which are contemplated to scavenge carcinogens).

Another form of modified tobacco is expanded or puffed tobacco. Includedherein are methods to produce reduced-exposure tobacco products byutilizing the tobacco provided herein, deproteinized tobacco fiber, andfreeze dried tobacco in any combination and in conjunction with expandedor puffed tobacco. More than 150 patents have been issued related totobacco expansion (e.g., U.S. Pat. No. 3,991,772, herein expresslyincorporated by reference in its entirety). “Expanded tobacco” is animportant part of tobacco filler which is processed through expansion ofsuitable gases so that the tobacco is “puffed” resulting in reduceddensity and greater filling capacity. It reduces the weight of tobaccoused in cigarettes. Advantageously, expanded tobacco reduces tar,nicotine and carbon monoxide deliveries and finds use, for example, inmaking low tar, low nicotine, and low carbon monoxide deliverycigarettes. Expanded tobacco is particularly useful in making low-tardelivery cigarettes. Carlton® cigarettes, which have had claims of beingthe lowest tar and nicotine delivery cigarette, are reportedly made witha very large percentage of expanded tobacco. However, use of expandedtobacco also results in reduced nicotine delivery, which can result incompensation.

Any method for expansion of tobacco known in the art can be used in themethods provided herein. The most common method used today incorporatesliquid carbon dioxide (U.S. Pat. Nos. 4,340,073 and 4,336,814, hereinexpressly incorporated by reference in its entirety). Liquid propane hasalso been used for making commercial cigarettes, predominantly in Europe(U.S. Pat. No. 4,531,529, herein expressly incorporated by reference inits entirety). Liquid propane offers advantages over carbon dioxidesince higher 3Q degrees of expansion are possible, in the range of 200%.Under pressure, the liquid carbon dioxide (or liquid propane) permeatesthe tobacco cell structure. When the tobacco is rapidly heated thecarbon dioxide (or liquid propane) expands the cell back to itspre-cured size.

Another form of modified tobacco is reconstituted tobacco. Includedherein are methods to produce reduced-exposure tobacco products byutilizing the tobacco provided herein, deproteinized tobacco fiber, andfreeze dried tobacco in any combination and in conjunction withreconstituted tobacco. “Reconstituted tobacco” (“Recon”) is an importantpart of tobacco filler made from tobacco dust and other tobacco scrapmaterial, processed into sheet form and cut into strips to resembletobacco. In addition to the cost savings, reconstituted tobacco is veryimportant for its contribution to cigarette taste from processing flavordevelopment using reactions between ammonia and sugars.

The process to produce sheets of Recon began during the 1950s. U.S.patents that describe such processes include: U.S. Pat. Nos. 3,499,454,4,182,349 4,962,774, and 6,761,175, herein expressly incorporated byreference in their entirety. Recon is traditionally produced fromtobacco stems and/or smaller leaf particles in a process that closelyresembles a typical paper making process. The tar and nicotine yields ofreconstituted tobacco are lower than those from equivalent quantities ofwhole tobacco leaf. This process entails processing the various tobaccoportions that are to be made into Recon. After the Recon sheets areproduced they are cut into a size and shape that resembles cut ragtobacco made from whole leaf tobacco. This cut Recon then gets mixedwith cut-rag tobacco and is ready for cigarette making. Cigarettes canbe manufactured with all Recon, no Recon, or any combination thereof.Most major brands have at least 10% of Recon in the Filler.

In another embodiment nicotine can be added, or nicotine salts, toproduce Recon, which is made from reduced-nicotine transgenic tobacco orany non-tobacco plant material including but not limited to herbalblends so that when the Recon is burned it yields substantially lesstobacco-specific nitrosamines and other carcinogens produced fromconventional cigarettes, yet satisfactory amounts are nicotine arepresent.

Processes of removing proteins from tobacco, thereby creating“deproteinized tobacco fiber” are known in the art, as exemplified inU.S. Pat. Nos. 4,289,147 and 4,347,324, herein expressly incorporated byreference in its entirety. Tobacco fiber is a major byproduct afterremoving protein. The fibrous remains from deproteinized tobacco can beincluded in any percentage as an ingredient of Recon. Cigarettes madefrom deproteinized tobacco have a different taste than conventionalcigarettes. However, appropriate amounts of additives, includingflavorings and nicotine, can be added to help alleviate this tastedeficiency.

Cigarettes containing deproteinized tobacco have a significant advantageover conventional cigarettes since they produce reduced levels ofcarcinogens and harmful combustion products. “A 71% reduction in proteincontent of a Flue-cured tobacco sheet resulted in an 81% reduction inthe TA98 Ames mutagenicity” of the pyrolytic condensate (Clapp, W. L.,et al., “Reduction in Ames Salmonella mutagenicity of cigarettemainstream smoke condensate by tobacco protein removal”, MutationResearch, 446, pg 167-174, 1999). Previous research in this area haddetermined that tobacco leaf protein might be the principal precursor ofmutagens in TSC (Matsumoto, et aI., “Mutagenicities of the pyrolysis ofpeptides and proteins”, Mutation Research, 56, pg 281-288, 1978).

Extracting tobacco fiber from genetically modified reduced-nicotinetobacco effectively eliminates virtually all carcinogenic TSNAs in suchtobacco, since nitrosamines require relatively high concentrations ofnicotine and other alkaloids to form at detectable levels. Therefore, itcan be advantageous to utilize reduced-nicotine tobacco inreduced-exposure cigarettes or other tobacco products to further reduceTSNAs. Nicotine can be either left out or introduced later in theprocess, which can also be in the form of nicotine salts.

PAHs are formed from high temperature pyrolysis of amino acids, sugars,paraffins, terpenes, phytosterols, celluloses and other components oftobacco. Most of these components are greatly reduced in tobacco fiber,effectively reducing formation of PAHs. Catechols and phenols,recognized carcinogenic co-factors in CS, would also be reduced sincelow levels of soluble sugar are present in tobacco fiber.

Harmful gas phase compounds such as hydrogen cyanide, nitrogen oxides,and carbon monoxide are also reduced when cigarette containing onlytobacco fiber is smoked compared to cigarettes made with whole-leaftobacco. Hydrogen cyanide is formed from burning proteins andchlorophyll. Nitrogen oxides are formed from burning soluble protein,chlorophyll, nitrates, and alkaloids. These components would not bepresent in significant amounts in deproteinized tobacco. Tobacco fiberhas approximately 85 percent less starches and cellulosic material thusreducing the major pyrolytic precursors of carbon monoxide.

In another embodiment, methods are provided to produce reconstitutedtobacco that includes extracted tobacco fiber derived from conventionaltobacco, reduced-nicotine transgenic tobacco, or increased-nicotinetransgenic tobacco.

If the tobacco curing process is circumvented, virtually no TSNAs willbe present in traditional tobacco products such as cigarettes, cigarfiller or wrapper, roll-your-own tobacco for cigarettes, pipe tobacco,chewing tobacco, snuff, reconstituted tobacco and other preparationsmade with freeze-dried tobacco would contain virtually no TSNAs sincetraditional curing processes are eliminated.

In another embodiment TSNAs can be virtually eliminated throughprocessing freshly harvested tobacco using lyophilization. This isaccomplished by processing freshly harvested tobacco throughfreeze-drying units located near tobacco farms. Tobacco processed inthis manner can be grown in a traditional fashion with spacing of plantsor in a biomass setting. In addition to the economic advantages ofeliminating the costs associated with the curing process, the tobaccocan now be grown in a biomass fashion that can create hundreds ofthousands of pounds of fresh tobacco per acre.

By growing tobacco in a biomass setting and immediately freeze dryingthe fresh tobacco for cigarettes, roll-your-own-tobacco, pipe tobacco,cigar filler or wrapper, chewing tobacco, snuff, and other versions ofsmokeless tobacco, labor is reduced not only by eliminating thetransplant of each plant from greenhouse to the field but also byeliminating traditional harvesting and curing of the tobacco. Also,farmland needed for this purpose is greatly reduced. The yield oftobacco from one acre of tobacco grown in biomass is equivalent toapproximately 100 acres of tobacco grown in a traditional manner.

“Tobacco biomass” is achieved by directly sowing an acre of land withcopious quantities of tobacco seed within a few inches of each other inthe field. Unlike tobacco planted with traditional spacing, individualplants can no longer be differentiated when tobacco is planted in abiomass fashion. An acre of tobacco biomass has the appearance of acontinuous, dense, green carpet. U.S. Pub. Pat. App. No. 20020197688,herein expressly incorporated by reference in its entirety, describessuch methods.

Lyophilization removes most of the water (−80%) from the weight of freshharvested tobacco biomass. The result is Freeze Dried Tobacco (“FDT”).FDT is easily pulverized into fine particles suitable for processinginto Recon. This Recon can be cut and made into any type of tobaccoproduct such as filler for cigarettes, roll-your-own-tobacco, pipetobacco, cigar filler or wrapper, chewing tobacco, snuff, and otherforms of smokeless tobacco. Flavorings and additives, including sugars,can be incorporated into the recon process.

Such Recon can be made from 100 percent FDT or in any proportion thatconsumers prefer. The lyophilization process can have adverse affects onthe taste of such tobacco products. Therefore, FDT can even be mixed inany percentage with traditional pulverized, cured tobacco so that themixture can be made into Recon. Alternatively, FDT can be mixed in anypercentages with any forms of traditional tobacco conducive formanufacturing cigarettes, roll-your-own tobacco, pipe tobacco, and cigarfiller or wrapper, chewing tobacco, snuff and other versions ofsmokeless tobacco in order to satisfy the tastes of the mass market.

In another embodiment, genetically modified reduced-nicotine tobacco canbe used for reducing TSNAs as described elsewhere herein, therebycreating an additional benefit of such cigarettes,roll-your-own-tobacco, pipe tobacco, cigar filler or wrapper, chewingtobacco, snuff and other versions of smokeless tobacco beingnon-addictive and without any TSNAs.

In another embodiment, nicotine can be added, in amounts that deliverthe desired physiological response, back to the FDT for uses incigarettes, cigar filler or wrapper, roll-your-own tobacco forcigarettes, pipe tobacco, chewing tobacco, snuff, and other versions ofsmokeless tobacco so that they will contain virtually no TSNAs.Cigarettes produced from tobacco fiber obtained from green leaf curedtobacco.

In another embodiment, Nicotiana rustica and/or increased-nicotinetransgenic Nicotiana tabacum are freeze dried after harvest and areincorporated into recon. The benefits are that the high alkaloid contentis preserved for low TNR cigarettes and that the tobacco curing step issaved. Also, the associated increase in TSNAs with high alkaloidtobaccos will not materialize. Preferred tobaccos for use with themethods described herein include genetically modified tobaccos asdescribed in the following sections.

Curing

The curing process, which typically lasts about 1 week, brings out theflavor and aroma of tobacco. Several methods for curing tobacco may beused, and indeed many methods have been previously disclosed. Forexample, U.S. Pat. No. 4,499,911 to Johnson; U.S. Pat. No. 5,685,710 toMartinez Sagrera; U.S. Pat. No. 3,905,123 to Fowler; U.S. Pat. No.3,840,025 to Fowler; and U.S. Pat. No. 4,192,323 to Home describeaspects of the tobacco curing process which may be used for someembodiments provided herein. Conventionally, “sticks” that are loadedwith tobacco are placed into bulk containers and placed into closedbuildings having a heat source known as a curing barn. A flue is oftenused to control the smoke (thus earning the term “Flue-cured”). Themethod of curing will depend, in some cases, on the type of tobacco-usecessation product desired, (i.e., snuff, cigarettes, or pipe tobacco maypreferably utilize different curing methods) and preferred methods mayvary from region to region and in different countries. In someapproaches, the stems and midveins of the leaf are removed from theleaves prior to curing to yield a high quality, low TSNA tobaccoproduct.

“Flue-curing” is a popular method for curing tobacco in Virginia, NorthCarolina, and the Coastal Plains regions of the United States. Thismethod is used mainly in the manufacture of cigarettes. Flue-curingrequires a closed building equipped with a system of ventilation and asource of heat. The heating can be direct or indirect (e.g., radiantheat). When heat and humidity are controlled, leaf color changes,moisture is quickly removed, and the leaf and stems dry. Carefulmonitoring of the heating and humidity can reduce the accumulation ofTSNAs.

Another curing method is termed “air-curing”. In this method, an openframework is prepared in which sticks of leaves (or whole plants) arehung so as to be protected from both wind and sun. Leaf color changesfrom green to yellow, as leaves and stems dry slowly.

“Fire-curing” employs an enclosed barn similar to that used forflue-curing. The tobacco is hung over low temperature fire so that theleaves cure in a smoke-laden atmosphere. This process uses lowertemperatures, so the process may take up to a month, in contrast toflue-curing, which takes about 6 to 8 days.

A further curing method, termed “sun-curing” is the drying of uncoveredsticks or strings of tobacco leaves in the sun. The best known sun-curedtobaccos are the so-called Oriental tobaccos of Turkey, Greece,Yugoslavia, and nearby countries.

The curing process, and most particularly the flue-curing process, isgenerally divided into the following four stages:

A) Firing Up: During this step, the tobacco leaves turn brightlemon-orange in color. This is achieved by a gradual increase intemperature.

B) Leaf Yellowing: In this step any moisture is removed. This createsthe “yellowing” of the tobacco. It also prepares the tobacco for dryingin the next step.

C) Leaf Drying: Leaf drying, an important step in the curing process,requires much time for the tobacco to dry properly. Additionally, airflow is increased in this step to facilitate the drying process.

D) Stem Drying: The drying process continues, as the stem of the tobaccoleaf becomes dried.

The cured tobacco may then be blended with other tobaccos or othermaterials to create the product to be used for the tobacco-use cessationmethod. The section below describes typical methods of blending andpreparing a tobacco product provided herein.

Tobacco Blending

It may be desirable to blend tobacco of varying nicotine levels tocreate the cessation product having the desired level of nicotine. Thisblending process is typically performed after the curing process, andmay be performed by conventional methods. Preferred tobacco blendingapproaches are provided below. In some embodiments, blending of thetransgenic tobacco is conducted to prepare the tobacco so that it willcontain specific amounts of nicotine, nornicotine, sterol and/or TSNA inspecific products. Preferably, the blending is conducted so that tobaccoproducts of varying amounts of nicotine are made in specific products.

A mixture that contains different types of tobacco is desirablysubstantially homogeneous throughout in order to avoid undesirablefluctuations in taste or nicotine levels. Typically, tobacco to beblended may have a moisture content between 30 and 75%. As an example,the tobacco is first cut or shredded to a suitable size, then mixed in amixing device, such as a rotating drum or a blending box. One such knownmixing device is a tumbling apparatus that typically comprises arotating housing enclosing mixing paddles which are attached to and,therefore, rotate with the housing to stir the tobacco componentstogether in a tumbling action as the drum turns.

After the desired tobaccos are thoroughly mixed, the resulting tobaccoblend is removed from the mixing apparatus and bulked to provide acontinuous, generally uniform quantity of the tobacco blend. The tobaccois then allowed to remain relatively undisturbed (termed the “bulkingstep”) for the required period of time before subsequent operations areperformed. The bulking step typically takes 30 minutes or less, and maybe carried out on a conveyor belt. The conveyor belt allows the blendedtobacco to remain in bulk form in an undisturbed condition while it iscontinuously moving the tobacco blend through the process from themixing stage to the expansion stage.

The tobacco blend is typically expanded by the application of steam. Thetobacco mixture is typically subjected to at least 0.25 pounds ofsaturated steam at atmospheric conditions per pound of blended tobaccofor at least 10 seconds to provide an increase in moisture of at least 2weight percent to the tobacco blend. After the tobacco blend has beenexpanded, it is dried. A typical drying apparatus uses heated air orsuperheated steam to dry the tobacco as the tobacco is conveyed by theheated air or steam stream through a drying chamber or series of dryingchambers. Generally, the wet bulb temperature of the drying air may befrom about 150 degrees F. to about 211 degrees F. The tobacco blend istypically dried to a moisture content of from about 60% to about 5%. Thedried, expanded tobacco blend is then in a suitable mode to be processedinto the tobacco-use cessation product as described below.

Some blending approaches begin with tobacco prepared from varieties thathave extremely low amounts of nicotine, nornicotine, sterols and/orTSNAs. By blending prepared tobacco from a low nicotine/TSNA variety(e.g., undetectable levels of nicotine and/or TSNAs) with a conventionaltobacco (e.g., Burley, which has 30,000 parts per million (ppm) nicotineand 8,000 parts per billion (ppb) TSNA; Flue-cured, which has 20,000 ppmnicotine and 300 ppb TSNA; and Oriental, which has 10,000 ppm nicotineand 100 ppb TSNA), tobacco products having virtually any desired amountof nicotine and/or TSNAs can be manufactured. Other approaches blendonly low nicotine/TSNA tobaccos (e.g., genetically modified Burley,genetically modified Virginia Flue-cured, and genetically modifiedOriental tobaccos that contain reduced amounts of nicotine and/or TSNAs)and/or low sterol tobaccos (e.g., Burley, Flue-cured, and Oriental).Tobacco products having various amounts of nicotine and/or TSNAs can beincorporated into tobacco-use cessation kits and programs to helptobacco users reduce or eliminate their dependence on nicotine andreduce the carcinogenic potential.

By one approach, a step 1 tobacco product is comprised of approximately25% low nicotine/TSNA tobacco and 75% conventional tobacco; a step 2tobacco product can be comprised of approximately 50% low nicotine/TSNAtobacco and 50% conventional tobacco; a step 3 tobacco product can becomprised of approximately 75% low nicotine/TSNA tobacco and 25%conventional tobacco; and a step 4 tobacco product can be comprised ofapproximately 100% low nicotine/TSNA tobacco and 0% conventionaltobacco. By another approach, a step 1 tobacco product is comprised ofapproximately 25% low sterol/PAH tobacco and 75% conventional tobacco; astep 2 tobacco product can be comprised of approximately 50% lowsterol/PAH tobacco and 50% conventional tobacco; a step 3 tobaccoproduct can be comprised of approximately 75% low sterol/PAH tobacco and25% conventional tobacco; and a step 4 tobacco product can be comprisedof approximately 100% low sterol/PAH tobacco and 0% conventionaltobacco. By another approach, a step 1 tobacco product is comprised ofapproximately 25% low sterol/PAH and low nicotine/TSNA tobacco and 75%conventional tobacco; a step 2 tobacco product can be comprised ofapproximately 50% low sterol/PAH and low nicotine/TSNA tobacco and 50%conventional tobacco; a step 3 tobacco product can be comprised ofapproximately 75% low sterol/PAH and low nicotine/TSNA tobacco and 25%conventional tobacco; and a step 4 tobacco product can be comprised ofapproximately 100% low sterol/PAH and low nicotine/TSNA tobacco and 0%conventional tobacco. A tobacco-use cessation kit can comprise an amountof tobacco product from any combination of the aforementioned blends tosatisfy a consumer for a single month program. That is, if the consumeris a one pack per day smoker, for example, a single month kit wouldprovide 7 packs from each step, a total of 28 packs of cigarettes. Eachtobacco-use cessation kit would include a set of instructions thatspecifically guide the consumer through the step-by-step process. Ofcourse, tobacco products having specific amounts of nicotine, TSNA,sterol and/or PAH would be made available in conveniently sized amounts(e.g., boxes of cigars, packs of cigarettes, tins of snuff, and pouchesor twists of chew) so that consumers could select the amount ofnicotine, TSNA, sterol and/or PAH they individually desire. There aremany ways to obtain various low nicotine/low TSNA and/or low sterol/lowPAH tobacco blends using the tobaccos and teachings described herein andthe following is intended merely to guide one of skill in the art to onepossible approach.

To obtain a step 1 tobacco product, which is a 25% low nicotine/TSNAblend, prepared tobacco from an approximately 0 ppm nicotine/TSNAtobacco can be mixed with conventional Burley, Flue-cured, or Orientalin a 25%/75% ratio respectively to obtain a Burly tobacco product having22,500 ppm nicotine and 6,000 ppb TSNA, a Flue-cured product having15,000 ppm nicotine and 225 ppb TSNA, and an Oriental product having7,500 ppm nicotine and 75 ppb TSNA. Similarly, to obtain a step 2product, which is 50% low nicotine/TSNA blend, prepared tobacco from anapproximately 0 ppm nicotine/TSNA tobacco can be mixed with conventionalBurley, Flue-cured, or Oriental in a 50%/50% ratio respectively toobtain a Burly tobacco product having 15,000 ppm nicotine and 4,000 ppbTSNA, a Flue-cured product having 10,000 ppm nicotine and 150 ppb TSNA,and an Oriental product having 5000 ppm nicotine and 50 ppb TSNA.Further, a step 3 product, which is a 75%/25% low nicotine/TSNA blend,prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco canbe mixed with conventional Burley, Flue-cured, or Oriental in a 75%/25%ratio respectively to obtain a Burly tobacco product having 7,500 ppmnicotine and 2,000 ppb TSNA, a Flue-cured product having 5,000 ppmnicotine and 75 ppb TSNA, and an Oriental product having 2,500 ppmnicotine and 25 ppb TSNA.

By a preferred method, conventional Virginia Flue-cured tobacco wasblended with genetically modified Burley (i.e., Burley containing asignificantly reduced amount of nicotine and TSNA) to yield a blendedtobacco that was incorporated into three levels of reduced nicotinecigarettes: a step 1 cigarette containing 0.6 mg nicotine, a step 2cigarette containing 0.3 mg nicotine, and a step 3 cigarette containingless than 0.05 mg nicotine. The amount of total TSNA was found to rangebetween approximately 0.17 μg/g-0.6 μg/g.

In some cigarettes, approximately, 28% of the blend was VirginiaFlue-cured tobacco, approximately 29% of the blend was geneticallymodified (i.e., reduced nicotine Burley), approximately 14% of the blendwas Oriental, approximately 17% of the blend was expanded Flue-curedstem, and approximately 12% was standard commercial Recon. The amount oftotal TSNAs in cigarettes containing this blend was approximately 1.5μg/g.

It should be appreciated that tobacco products are often a blend of manydifferent types of tobaccos, which were grown in many different parts ofthe world under various growing conditions. As a result, the amount ofnicotine, TSNAs, sterols and PAHs will differ from crop to crop.Nevertheless, by using conventional techniques one can easily determinean average amount of nicotine, TSNA, sterol and PAH per crop used tocreate a desired blend. It should also be appreciated thatreconstituted, expanded, chemically treated, or microbial treatedtobacco can be blended with the modified tobacco described herein, suchas, for example the transgenic tobacco described herein. By adjustingthe amount of each type of tobacco that makes up the blend one of skillcan balance the amount of nicotine, TSNA, sterol and/or PAH with otherconsiderations such as appearance, flavor, and smokability. In thismanner, a variety of types of tobacco products having varying level ofnicotine, TSNA, sterol and/or PAH, as well as, appearance, flavor andsmokability can be created.

A. Genetically Modified Tobacco

In some embodiments, the modified tobacco is a genetically modifiedtobacco. Several approaches to create genetically modified tobaccohaving a reduced amount of a harmful compound are described. Manyembodiments concern nucleic acid constructs that inhibit the expressionof a gene, which regulates production of a compound that is associatedwith a tobacco-related disease. Since these nucleic acid constructsefficiently reduce the presence of a compound that contributes to atobacco-related disease, the genetically modified tobacco, prepared asdescribed herein, can be used to create a tobacco product, such as acigarette, snuff or pipe tobacco, which has a reduced potential tocontribute to a tobacco-related disease. That is, embodiments providedherein concern reduced risk tobacco products made from reduced risktransgenic tobacco created using the nucleic acid constructs describedherein.

More specifically, embodiments provided herein concern nucleic acidconstructs that inhibit the expression of a number of genes involved inthe synthesis and regulation of the production of nicotine, nornicotine,and/or sterols in tobacco. Alkaloids such as nicotine and nornicotineare precursors for a number of harmful compounds that contribute totobacco-related disease (e.g., the tobacco specific nitrosamines(TSNAs): N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT),N′-nitrosoanabasine (NAB),4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal(NNA)-4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL),4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and/or4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid (iso-NNAC) andacrolein). Sterols are precursors for a number of harmful compounds,which are generated by pyrolysis of tobacco, that also contribute totobacco-related disease (e.g., polyaromatic hydrocarbons (PAHs), such asbenz[a]pyrene (BAP), heterocyclic hydrocarbons, terpenes, paraffins andaromatic amines). Because the presence of these harmful compounds intobacco contributes to tobacco-related disease, a transgenic orgenetically modified tobacco that comprises a reduced amount of any oneof these compounds, as compared to a reference tobacco has a reducedpotential to contribute to a tobacco-related disease.

Other embodiments concern nucleic acid constructs for heterologousexpression of a gene that reduces, or is related to production of acompound that reduces, the harmful effect of one or more compoundsassociated with a tobacco-related disease. Since these nucleic acidconstructs introduce or increase the presence of a compound that resultsin reduction of the harmful effect of a compound associated with atobacco-related disease, the genetically modified tobacco, prepared asdescribed herein, can be used to create a tobacco product, such as acigarette, snuff or pipe tobacco, which has a reduced potential tocontribute to a tobacco-related disease. That is, embodiments providedherein concern reduced risk tobacco products made from reduced risktransgenic tobacco created using the nucleic acid constructs describedherein.

Other embodiments are directed to genetically modified tobacco in whichexpression of two or more genes in the biosynthetic pathway of acompound associated with a tobacco-related disease is inhibited.Inhibition of two or more genes in the biosynthetic pathway of acompound associated with a tobacco-related disease can be attained byinhibition of two or more genes that act on a substrate at the same stepin the biosynthetic pathway (e.g., inhibition of two or more isoforms ofa biosynthetic gene) or inhibition of two or more genes that act on asubstrate at different steps in the biosynthetic pathway. In suchembodiments, the genetically modified tobacco can contain one or moreheterologous nucleic acids such as the nucleic acids and constructsprovided herein, where the heterologous nucleic acids can contain one ormore sequences that can inhibit expression of two or more genes in thebiosynthetic pathway of a compound associated with a tobacco-relateddisease.

Other embodiments are directed to genetically modified tobacco in whichthe active form of a gene in the biosynthetic pathway of a compoundassociated with a tobacco-related disease is inhibited. The active formof a gene in the biosynthetic pathway of a compound associated with atobacco-related disease can be inhibited by any of a variety of methodsfor inhibiting protein activity, including, but not limited to: knockingout part or all of a gene encoding the endogenous protein using, forexample, homologous recombination; and heterologous expression of adominant negative protein that inhibits the activity of the endogenousprotein.

By using the constructs described herein, the amount of harmfulcompounds in tobacco or the harmful effects thereof, such as alkaloidsand sterols, can be reduced or removed and a tobacco product comprisingthis genetically modified tobacco, with or without exogenous nicotine,will have a reduced potential to contribute to a tobacco-relateddisease. That is, genetically modified tobacco comprising the constructsdescribed herein can be used to manufacture “reduced risk” tobaccoproducts (e.g., a tobacco product comprising a reduced endogenousnicotine, reduced endogenous nornicotine, and/or reduced steroltobacco), such as a cigarette, snuff or pipe tobacco, which may haveexogenous nicotine incorporated therein.

Accordingly, embodiments provided herein concern genetically modifiedtobacco and tobacco products containing a tobacco that comprises agenetic modification, which have a reduced amount or are substantiallyfree of a harmful compound including, but not limited to, nicotine,nornicotine, a sterol, an acrolein, an aldehyde, a TSNA selected fromthe group consisting of N′-nitrosonornicotine (NNN),4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),N′-nitrosoanatabine (NAT), and/or N′-nitrosoanabasine (NAB) or generatea reduced amount of a PAH, a BAP, a heterocyclic hydrocarbon, anaromatic amine upon pyrolysis, wherein this reduced risk geneticallymodified tobacco is made by lowering the expression of a gene in saidtobaccos with one of the constructs described herein. Preferredembodiments include a transgenic tobacco and a tobacco product (e.g.,cigarette) that comprises a cured tobacco comprising a geneticmodification and comprising or delivering by FTC method a reduced amountof nicotine or total alkaloid (e.g., below a conventional level ofnicotine or total alkaloid typical for the strain of plant, preferably,less than or equal to 3,000 ppm, 2000 ppm, 1000 ppm, or 500 ppm),wherein said genetic modification comprises an inhibition of a gene thatregulates the production of nicotine and/or nornicotine, such asarginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADHdehydrogenase, omithine decarboxylase (ODC), phosphoribosylanthranilateisomerase (PRAI), putrescine N-methyltransferase (PMT), quinolatephosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase(SAMS), or A622 or comprises an inhibition of a gene that regulates theproduction of sterol biosynthesis include HMG-CoA reductase, 14alphademethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase,A8-A7-isomerase, or C4-demethylase, using one or more of the constructsdescribed herein.

Preferred embodiments also include a transgenic tobacco and a tobaccoproduct (e.g., cigarette, snuff or pipe tobacco) that comprises a curedtobacco comprising a genetic modification and a reduced amount of asterol (e.g., comprises an amount of sterol or delivers and amount ofsterol that is below a conventional level of said sterol typical for thestrain of plant) wherein said genetic modification comprises aninhibition of a gene that regulates the production of a sterol intobacco using one or more of the constructs described herein. Relatedembodiments include a transgenic tobacco and tobacco product madetherefrom (e.g., a cigarette, snuff or pipe tobacco) that upon pyrolysisgenerates a reduced amount of a PAH, BAP, a heterocyclic hydrocarbon, oran aromatic amine, as compared to that generated by a reference tobaccoor reference tobacco product (e.g., IM16, 2R4F or 1R5F), a commerciallyavailable tobacco product of the same class (e.g., full-flavor, lights,and ultra-lights), or, preferably, a tobacco of the same variety (e.g.,Burley, Virginia Flue-cured, or Oriental) or strain (e.g., LA Burley 21,K326, Tn90, Djebe1174) as the transgenic tobacco prior to geneticmodification).

Preferred embodiments also include a transgenic tobacco and a tobaccoproduct (e.g., cigarette, snuff or pipe tobacco) that comprises a curedtobacco comprising a genetic modification and a reduced amount ofnicotine or total alkaloid and a sterol (e.g., comprise or provides anamount of nicotine or total alkaloid an/or sterols that is below aconventional level of nicotine, total alkaloid, or sterol typical forthe strain of plant) wherein said genetic modification comprises aninhibition of a gene that regulates the production of both nicotine andsterols in tobacco. That is, embodiments provided herein concernisolated nucleic acids, isolated nucleic acid cassettes, and isolatednucleic acid constructs that inhibit the expression of a plurality ofgenes that regulate the production of nicotine and TSNAs, isolatednucleic acids, isolated nucleic acid cassettes, and isolated nucleicacid constructs that inhibit the expression of a plurality of genes thatregulate the production of sterols and, thus PAHs, and isolated nucleicacids, isolated nucleic acid cassettes, and isolated nucleic acidconstructs that inhibit the expression of a plurality of genes thatregulate the production of nicotine and TSNAs and sterols and, thus,PAHs (e.g., a double knock-out of at least two different genes thatregulate the production of at least two different harmful compounds intobacco).

In some embodiments, the tobacco that is substantially free or comprisesa reduced amount of nicotine, nornicotine, TSNAs, sterols, and/orproduces a reduced amount of PAHs upon pyrolysis is made by exposing atleast one tobacco cell of a selected variety (e.g., Burley, VirginiaFlue-cured, or Oriental) to an exogenous nucleic acid construct encodingan interfering RNA comprising an RNA duplex that comprises a firststrand having a sequence that is substantially similar or identical toat least a portion of the coding sequence of a target gene and/or targetgene product involved in nicotine biosynthesis or sterol biosynthesis,and a second strand that is complementary or substantially complementaryto the first strand. In some embodiments, the nucleic acid constructfurther comprises a nucleotide sequence encoding the interfering RNAoperably linked to a promoter operable in a plant cell. The tobacco cellis transformed with the nucleic acid construct, transformed cells areselected and at least one transgenic tobacco plant is regenerated fromthe transformed cells. The transgenic tobacco plants described hereincan contain a reduced amount of anyone of nicotine, nornicotine, TSNAsand/or a sterol as compared to a control tobacco plant of the samevariety. In some embodiments, nucleic acid constructs encodinginterfering RNAs (RNAi) comprising a first strand having a sequencesubstantially similar or identical to the entire coding sequence of atarget gene and/or target gene product involved in nicotine or sterolbiosynthesis, and a second strand that is complementary or substantiallycomplementary to the first strand, are contemplated.

In some embodiments, the genetically modified tobacco provided hereinwill be genetically stable for at least 2, 3, 4, 5, 6, 8, 10, 12, 15,20, 25, 30, 40 or 50, or more, generations. For example, the geneticallymodified tobacco produces a reduced amount of a compound associated witha tobacco related disease for at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20,25, 30, 40 or 50, or more, generations. It is contemplated, for example,that crossings of multiple tobaccos each having different geneticmodifications that are stable over many generations can be performed soas to obtain a genetically modified tobacco having a reduced level ofexpression of a plurality of genes that encode precursors for varioustobacco related diseases.

In some embodiments, the genetically modified tobacco provided hereinwill have agronomic characteristics suitable for commercial production.Although in some instances genetically modified tobacco can haveagronomic characteristics that are different from conventional tobacco,such a tobacco can be suitable for commercial production because thesedifferent agronomic characteristics can be compensated for by employingtechniques common to those of skill in the art. That is, although theagronomic characteristics for a genetically modified tobacco created asdescribed herein may differ from those of conventional tobacco, suchalterations may not necessarily yield a plant that is no longer suitablefor commercial production. For example, a genetically modified tobaccomay have a reduced root mass, but tobacco plants having reduced rootmass can nevertheless be suitable for commercial production when suchtobaccos are raised under conditions in which the plants are thoroughlyirrigated and/or not subjected to drought conditions. Additionalnutritional requirements (e.g., nitrogen) may be required. Any of avariety of conventional agronomic methods can be used to producecommercial quantities of a genetically modified tobacco, where suchmethods include, but are not limited to, irrigation, fertilization,providing nutrients for plant growth, and use of pesticides. As referredto herein, a genetically modified tobacco that is suitable forcommercial production is a genetically modified tobacco that, underappropriate agronomic conditions will produce at least 25%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, tobacco useful forcreation of a tobacco product relative to an unmodified or conventionaltobacco, grown under its standard growing conditions.

1. Genes to Modify

In some embodiments, the gene product is one that is involved innicotine biosynthesis. Such enzymes include, but are not necessarilylimited to, putrescene N-methyltransferase (PMTase), N-methylputresceneoxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADHdehydrogenase, phosphoribosylanthranilate isomerase and quinolatephosphoribosyl transferase (QPTase). In preferred embodiments, the geneproduct that is inhibited using a construct described herein is QPTase,PMTase, and A622. In some embodiments, the tobacco that is madesubstantially free of nicotine and/or TSNAs (e.g., comprises or deliversless than or equal to 0.5 mg/g nicotine and/or less than or equal to 0.5μg/g collective content of NNN, NAT, NAB, and NNK) is prepared from avariety of Burley tobacco (e.g., Burley 21 or Tn90), Oriental tobacco(Djebal 174), or Virginia Flue-cured (K326) tobacco. It should beunderstood, however, that most tobacco varieties can be made to havereduced amounts of nicotine and/or TSNAs or can be made substantiallyfree of nicotine and/or TSNAs by using the embodiments described herein.For example, plant cells of the variety Burley 21 are used as the hostfor the genetic engineering that results in the reduction of nicotineand/or TSNAs so that the resultant transgenic plants are a Burley 21variety that has a reduced amount of nicotine and/or TSNAs.

Accordingly, some embodiments concern a tobacco that comprises a geneticmodification comprising a reduced amount or a reduced level ofexpression of QPTase, PMTase, or A622, comprising or delivering areduced amount of nicotine or total alkaloid and/or a collective contentof TSNA (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 0.5 μg/g(e.g., 0.05 μg/g, 0.1 μg/g, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g).More embodiments concern a tobacco that comprises or delivers a reducedamount or a reduced level of expression of A622, a normal orconventional amount of nicotine (e.g., comprising or delivering by FTCmethodology an amount of nicotine equal to, less than, or greater than0.9 mg/g, 1.0 mg/g, 1.1 mg/g, 1.2 mg/g, 1.3 mg/g, 1.4 mg/g, 1.5 mg/g,1.6 mg/g, 1.7 mg/g, 1.8 mg/g, 1.9 mg/g, and 2.0 mg/g), and a reducedamount of nornicotine (e.g., comprising or delivering by FTC methodologyan amount of nornicotine less than or equal to 0.5 μg/g), and/or areduced amount of NNN (e.g., comprising or delivering by FTC methodologyan amount of total TSNAs equal to or less than 0.05 μg/g, 0.1 μg/g, 0.2μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g). That is, particular lines oftransgenic tobacco containing the A622 inhibition cassette describedherein were unexpectedly found to have a reduced level of nornicotinebut conventional levels of nicotine. This finding is particularlyimportant since nornicotine may be a more important precursor for NNNthan nicotine. (See Carmella et al., Carcinogenesis, Vol. 21, No. 4,839-843, (April 2000), herein expressly incorporated by reference in itsentirety). In other transgenic lines, wherein the A622 gene wasinhibited using one of the constructs described herein, it was foundthat both nicotine and nornicotine were effectively reduced (e.g., totalalkaloids were less than or equal to 7,000 ppm, 5000 ppm, 3000 ppm, 1000ppm, or 500 ppm).

Some of the nucleic acid constructs provided herein employ interferingRNAs (e.g., siRNAs or dsRNAs) that comprise an RNA duplex wherein eachRNA portion of the duplex is at least, greater than, or equal to 30, 40,50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280,300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560,580, 600, 620, 640, 660, 680, 700, 750, 1000, 1500, 2000, 2500, or 5000consecutive nucleotides complementary or substantially complementary toan mRNA that encodes a gene product or the entire coding sequence of theenzyme or complement thereof of an enzyme that regulates nicotine orsterol biosynthesis. In some embodiments, the RNA duplex comprises afirst RNA strand that is complementary to an mRNA that encodes a geneproduct involved in nicotine or sterol biosynthesis and a second RNAstand that is complementary to said first strand. Some interfering RNAsprovided herein can comprise two separate RNA strands hybridized to eachother by hydrogen bonding. Other interfering RNAs comprise a single RNAstrand comprising a first and second regions of nucleotide sequence thatare complementary to each other. In such embodiments, the first andsecond regions of nucleotide sequence are separated by a nucleotidesequence (e.g., a “linker”) that permits or, in the case of the FAD2intron described herein, facilitates formation of a hairpin structureupon hybridization of the first and second regions. This “linker” thatpermits formation of a hairpin structure is preferably at least, greaterthan, or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460,480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900,1000 or more nucleotides in length.

A preferred method of producing tobacco having a reduced amount ofnicotine and TSNAs, involves genetic engineering directed at reducingthe levels of nicotine and/or nornicotine or other alkaloids. Any enzymeinvolved in the nicotine synthesis pathway can be a suitable target forgenetic engineering to reduce levels of nicotine and, optionally, levelsof other alkaloids including nornicotine. Suitable targets for geneticengineering to produce tobacco having a reduced amount of nicotineand/or nitrosamines, especially TSNAs, include but are not limited toputrescene N-methyltransferase, N-methylputrescene oxidase, ornithinedecarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase,phosphoribosylanthranilate isomerase, quinolate phosphoribosyltransferase (QPTase) or a combination of any of the above targets.Additionally, enzymes that regulate the flow of precursors into thenicotine or sterol synthesis pathway are suitable targets for geneticengineering to produce tobacco with a reduced amount of nicotine andnitrosamines, especially TSNAs, and tobaccos with reduced amounts ofsterols, which produce a reduced amount of PAHs upon pyrolysis. Suitablemethods of genetic engineering are known in the art and include, forexample, the use of antisense and sense suppression technology to reduceor eliminate the production of enzymes, the use of interfering RNAmolecules (gene silencing) as described herein to reduce or eliminatethe expression of gene products, and the use of random or targetedmutagenesis to disrupt gene function, for example, using T-DNA insertionor EMS mutagenesis. The next section provides more description of thesetechniques.

2. Modification Methods

a) Knockouts

Inhibition of Gene Expression Using Nucleic Acids

Inhibition of gene expression refers to the absence or observablereduction in the level of polypeptide and/or mRNA gene product. Someembodiments provided herein relate to inhibiting the expression of oneor more genes involved in the biosynthesis of nicotine, nornicotine,and/or sterols by genetically modifying a plant cell, such as a tobaccocell, by providing the cell with an inhibitory nucleic acid that reducesor eliminates the production of a gene product involved in nicotine orsterol biosynthesis. Inhibitory nucleic acids include, but are notlimited to, interfering RNAs, antisense nucleic acids and catalyticRNAs. Some preferred embodiments provided herein relate to interferingRNAs (RNAi).

RNA interference and gene silencing are terms that are used to describea phenomenon by which the expression of a gene product is inhibited byan interfering RNA molecule. Interfering RNA molecules aredouble-stranded RNAs (dsRNA) that are expressed in or otherwiseintroduced into a cell. The dsRNA molecules may be of any length,however, short dsRNA constructs are commonly used. Such constructs areknown as small interfering RNAs (siRNA), and are typically 21-23 bp inlength.

RNA interference is exhibited by nearly every eukaryote and is thoughtto function by a highly conserved mechanism (Dillin, A. PNAS,100:6289-91). As with antisense inhibition of gene expression,inhibition mediated by RNA interference is gene specific. However, incontrast to antisense-mediated inhibition, inhibition mediated byinterfering RNA appears to be inherited (Dillin, A. PNAS, 100:6289-91).Without being bound by theory, it is believed that specificity isachieved through nucleotide sequence interaction between complementaryportions of a target mRNA and the interfering RNA. The target mRNA isselected based on the specific gene to be silenced. In particular, thetarget mRNA, corresponds to the sense strand of the gene to be silenced.An interfering RNA, such as a dsRNA or an siRNA, comprises an RNAduplex, which includes a first strand that is substantially similar oridentical to at least a portion of the nucleotide sequence of the targetmRNA, and a second strand having a nucleotide sequence that iscomplementary or substantially complementary to the first strand.

When used herein with reference to an RNA duplex of the interfering RNA,it will be appreciated that the terms “first strand” and “second strand”are used in a relative sense. For example, the first strand of an RNAduplex can be selected to comprise either a nucleotide sequencesubstantially similar or identical to at least a portion of thenucleotide sequence of the target mRNA or a nucleotide sequence that iscomplementary or substantially complementary to at least a portion ofthe nucleotide sequence of the target mRNA. If the first strand isselected to be substantially similar or identical to at least a portionof the nucleotide sequence of the target mRNA, then the second strandwill be complementary to at least a portion of the target mRNA becauseit is complementary to the first strand. If the first strand is selectedto be complementary or substantially complementary to at least a portionof the target mRNA, then the second strand will be substantially similaror identical to at least a portion of the nucleotide sequence of thetarget mRNA because it is complementary to the first strand.

As used herein with reference to nucleic acids, “portion” means at least5 consecutive nucleotides, at least 6 consecutive nucleotides, at least7 consecutive nucleotides, at least 8 consecutive nucleotides, at least9 consecutive nucleotides, at least 10 consecutive nucleotides, at least11 consecutive nucleotides, at least 12 consecutive nucleotides, atleast 13 consecutive nucleotides, at least 14 consecutive nucleotides,at least 15 consecutive nucleotides, at least 16 consecutivenucleotides, at least 17 consecutive nucleotides, at least 18consecutive nucleotides, at least 19 consecutive nucleotides, at least20 consecutive nucleotides, at least 21 consecutive nucleotides, atleast 22 consecutive nucleotides, at least 23 consecutive nucleotides,at least 24 consecutive nucleotides, at least 25 consecutivenucleotides, at least 30 consecutive nucleotides, at least 35consecutive nucleotides, at least 40 consecutive nucleotides, at least45 consecutive nucleotides, at least 50 consecutive nucleotides, atleast 60 consecutive nucleotides, at least 70 consecutive nucleotides,at least 80 consecutive nucleotides, at least 90 consecutivenucleotides, at least 100 consecutive nucleotides, at least 125consecutive nucleotides, at least 150 consecutive nucleotides, at least175 consecutive nucleotides, at least 200 consecutive nucleotides, atleast 250 consecutive nucleotides, at least 300 consecutive nucleotides,at least 350 consecutive nucleotides, at least 400 consecutivenucleotides, at least 450 consecutive nucleotides, at least 500consecutive nucleotides, at least 600 consecutive nucleotides, at least700 consecutive nucleotides, at least 800 consecutive nucleotides, atleast 900 consecutive nucleotides, at least 1000 consecutivenucleotides, at least 1200 consecutive nucleotides, at least 1400consecutive nucleotides, at least 1600 consecutive nucleotides, at least1800 consecutive nucleotides, at least 2000 consecutive nucleotides, atleast 2500 consecutive nucleotides, at least 3000 consecutivenucleotides, at least 4000 consecutive nucleotides, at least 5000consecutive nucleotides or greater than at least 5000 consecutivenucleotides. In some preferred embodiments, a portion of a nucleotidesequence is between 20 and 25 consecutive nucleotides. In otherpreferred embodiments, a portion of a nucleotide sequence is between 21and 23 consecutive nucleotides. In some embodiments provided herein, aportion of a nucleotide sequence includes the full-length codingsequence of the gene or the target mRNA.

Some preferred interfering RNAs that are described herein comprise anRNA duplex, which comprises a nucleotide sequence that is substantiallysimilar or identical to at least a portion of the coding strand of agene involved in nicotine or sterol biosynthesis. Although nucleic acidsequences that are substantially similar or identical to at least aportion of the coding strand of the target gene involved in nicotinebiosynthesis are preferred, it will be appreciated that nucleotidesequences with insertions, deletions, and single point mutationsrelative to the target sequence are also effective for inhibition ofgene expression. Sequence identity may be determined by sequencecomparison and alignment algorithms known in the art (see Gribskov andDevereux, Sequence Analysis Primer, Stockton Press, 1991, and referencescited therein) and calculating the percent difference between thenucleotide sequences by, for example, the Smith-Waterman algorithm asimplemented in the BESTFIT software program using default parameters(e.g., University of Wisconsin Genetic Computing Group). Greater than90% sequence identity, or even 100% sequence identity, between theinterfering RNA and a portion of the target gene is preferred. Inespecially preferred embodiments, at least about 21 to about 23contiguous nucleotides in the target gene are greater than 90% identicalto a sequence present in the interfering RNA.

In other embodiments provided herein, the duplex region of the RNA maybe defined functionally as including a nucleotide sequence that iscapable of hybridizing with a portion of the target gene transcript.Exemplary hybridization conditions are 400 mM NaCl, 40 mM PIPES pH 6.4,1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing.

The modification of nicotine levels in tobacco plants by antisenseregulation of putrescene methyl transferase (PMTase) expression has beenproposed in U.S. Pat. Nos. 5,369,023 and 5,260,205, to Nakatani andMalik, and in PCT application WO 94/28142 and U.S. Pat. No. 5,668,295 toWahad and Malik, which describe DNA encoding PMT and the use of senseand antisense PMT constructs, the entire disclosures of each of whichare hereby expressly incorporated by reference in their entireties.Other genetic modifications proposed to reduce nicotine levels aredescribed in PCT application WO 00/67558, to Timko, and WO 93/05646, toDavis and Marcum; the entire contents of each are hereby expresslyincorporated by reference in their entireties. Although theseinvestigators made significant contributions, there were significantdrawbacks to their experimental design.

Provided herein are tobacco and tobacco products in which a plurality ofgenes involved in nicotine biosynthesis are inhibited. Most notably, itis presently revealed that there are several different PMT genes andeach may play a role in nicotine biosynthesis. Knocking-out only one PMTgene can create a leaky system allowing the other PMT genes tocompensate for the reduction. Accordingly, each of the PMT constructsdescribed herein were designed to inhibit a plurality of different PMTgenes with a single construct. That is, the PMT constructs describedherein are designed to complement common regions to all five of the PMTgenes so that inhibition of each of the PMT genes can be accomplishedwith one inhibitory fragment. Although many of the approaches describedin this section have significant drawbacks, it should be understood thatany or all of these techniques can be used with other techniques, asdescribed herein, to make tobacco and tobacco products having reducednicotine.

In some embodiments that employed the A622 inhibition construct, it wasfound that transgenic tobacco that had conventional levels of nicotinebut significantly reduced levels of nornicotine were produced. Thisparticular line of tobacco is particularly useful because nornicotinemay be the most significant precursor for NNN in tobacco. Accordingly,reduced risk conventional cigarettes and other tobacco products (e.g.,snuff) comprising the A622 inhibition construct are embodiments.

As described above, interfering RNAs disclosed herein comprise asequence that is complementary to at least a portion of the sense strandof a gene encoding a target mRNA, which produces a polypeptide that isinvolved in nicotine biosynthesis. Preferred targets are the products ofthe quinolate phosphoribosyltransferase (QTPase) gene, the putresceneN-methyltransferase (PMTase) gene, and the A622 gene. However, it willbe appreciated that interfering RNAs specific for other gene products orcombinations of gene products involved in nicotine and nornicotinebiosynthesis and/or sterol biosynthesis are contemplated. For example,additional gene products involved in nicotine biosynthesis include, butare not limited to, N-methylputrescene oxidase, ornithine decarboxylase,S-adenosylmethionine synthetase, NADH dehydrogenase, andphosphoribosylanthranilate isomerase. Additionally, it will beappreciated that interfering RNAs specific for other gene products orcombinations of gene products involved in sterol biosynthesis includeHMG-CoA reductase, 14alpha demethylase, squalene synthase, SMT2, SMT1,C14 sterol reductase, A8-A7-isomerase, and C4-demethylase.

Additionally, the interfering RNAs described herein can comprise aplurality nucleotide sequences that are each complementary to differentportions of the sense strand of a gene involved in nicotine and/orsterol biosynthesis. Alternatively, the interfering RNAs describedherein can comprise a plurality nucleotide sequences that are eachcomplementary to at least a portion of the sense strands of differentgenes involved in nicotine and/or sterol biosynthesis. Still further, asingle RNAi construct or inhibition cassette can be used to inhibit aplurality of genes involved in the regulation of the production ofnicotine, nornicotine, or sterols. For example, as described below, itwas found that the A622 inhibitory fragment and inhibition cassette(SEQ. ID. Nos. 5 and 26) efficiently reduced production of nicotine andnornicotine in some lines of tobacco and in other lines of tobaccoconventional levels of nicotine were maintained but the amount ofnornicotine in said tobacco was 0.00 mg/g. Still further, the PMTaseinhibitory sequence and PMTase inhibition cassette (SEQ. ID. Nos. 4 and25) were designed to complement common regions of a plurality of PMTasegenes so that the production of multiple gene products can be inhibitedor reduced with a single construct.

In still more embodiments, it is contemplated that a single T-DNAcontaining construct be used to overexpress one gene and, in the sameconstruct, inhibiting expression of a second gene. That is, someembodiments concern constructs, tobacco containing said constructs, andtobacco products containing said tobacco, wherein said constructscomprise an overexpression cassette that comprises a gene that regulatesthe production of a compound that improves the composition of thetobacco (e.g, overexpression of a gene encoding an antioxidant) and, onthe same construct, an inhibition cassette that comprises an inhibitorysequence that reduces the production of a compound that contributes to atobacco related disease (e.g., nicotine, nornicotine, or a sterol).

In preferred embodiments, the interfering RNAs described herein compriseat least one region of double-stranded RNA (duplex RNA). This duplex RNAcan range from about 10 bp in length to about 10,000 bp in length. Insome embodiments, the duplex RNA ranges from about 15 bp in length toabout 1500 bp in length. In other embodiments, the duplex RNA rangesfrom about 20 bp in length to about 1200 bp in length. In still otherembodiments, the duplex RNA ranges from about 21 bp in length to about23 bp in length. In a preferred embodiment, the duplex RNA has a lengthof 22 bps. Short regions of duplex RNA are often designated siRNA,whereas longer regions of RNA duplex are often termed dsRNA. In someembodiments provided herein, the interfering RNA duplex region is adsRNA. In other embodiments, the interfering RNA duplex region is ansiRNA. In a preferred embodiment, the duplex region about the length ofthe coding sequence of a target mRNA encoding a polypeptide involved innicotine biosynthesis.

Interfering RNAs described herein can be generated using a variety oftechniques. For example, an interfering RNA can be generated in a hostcell in vivo by providing the cell with one or more a nucleic acidconstructs that comprise the nucleic acids necessary to encode thestrands of a double-stranded RNA. Such constructs can be included invarious types of vectors. Exemplary vectors contemplated herein include,but are not limited to, plasmids, viral vectors, viroids, replicable andnonreplicable linear DNA molecules, replicable and nonreplicable linearRNA molecules, replicable and nonreplicable circular DNA molecules andreplicable and nonreplicable circular RNA molecules. Preferred vectorsinclude plasmid vectors, especially vector systems derived from theAgrobacterium Ti plasmid, such as pCambia vectors and derivativesthereof.

In some embodiments, both strands of the double-stranded region of theinterfering RNA can be encoded by a single vector. In such cases, thevector comprises a first promoter operably linked to a first nucleicacid which is substantially similar or identical to at least a portionof the target mRNA. The vector also comprises a second promoter operablylinked to a second nucleic acid, which is complementary or substantiallyto the first nucleic acid.

Another type of single vector construct, which can be used to generateinterfering RNA, encodes a double-stranded RNA hairpin. In suchembodiments, the vector comprises a promoter operably linked to anucleic acid that encodes both strands of the duplex RNA. The firstnucleotide sequence, which encodes the strand that is substantiallysimilar or identical to at least a portion of the target mRNA, isseparated from the second nucleotide sequence, which encodes a strandcomplementary or substantially complementary to the first strand, by aregion of nucleotide sequence that does not substantially hybridize witheither of the strands. This nonhybridizing region permits the RNAsequence transcribed from the vector promoter to fold back on itself,thereby permitting the complementary RNA sequences to hybridize so as toproduce an RNA hairpin. Vectors comprising a plurality of nucleic acids,each of which encode both strands of the duplex RNA are alsocontemplated.

Other embodiments provided herein relate to multiple vector systems forthe production of interfering RNA. In one example, a multiple vectorsystem is used to produce a single interfering RNA that is specific fora single gene product involved in nicotine biosynthesis. In suchembodiments, at least two vectors are used. The first vector comprises apromoter operably linked to a first nucleic acid that encodes a firststrand of the RNA duplex that is present in the interfering RNA. Thesecond vector comprises a promoter operably linked to a second nucleicacid that encodes the second strand of the RNA duplex, which iscomplementary to the first strand.

Other multiple vector systems are combinations of vectors, wherein eachvector in the system encodes a different interfering RNA. Each of theinterfering RNAs is specific for different gene products involved innicotine biosynthesis. In some embodiments, the vectors in a multiplevector system can encode different interfering RNAs that are specific todifferent portions of a single gene product involved in nicotinebiosynthesis.

It will be appreciated that the promoters used in the above-describedvectors can either be constitutive or regulated. Constitutive promotersare promoters that are always expressed. The constitutive promotersselected for use in the above-described vectors can range from weakpromoters to strong promoters depending on the desired amount ofinterfering RNA to be produced. Regulated promoters are promoters forwhich the desired level of expression can be controlled. An example of aregulated promoter is an inducible promoter. Using an inducible promoterin the above-described vector constructs permits expression of a widerange of concentrations of interfering RNA inside a cell.

It will also be appreciated that there is no requirement that the sameor same types of promoters be used in vectors or multiple vector systemsthat comprise a plurality of promoters. For example, in some vectors orvector systems, a first promoter, which controls the expression of thefirst interfering RNA strand, can be an inducible promoter, whereas thesecond promoter, which controls the expression of the second RNA strand,can be a constitutive promoter. This same principal can also beillustrated in a multiple vector system. For example, a multiple vectorsystem may have three vectors each of which includes one or moredifferent types of promoters. Such a system can include, for example, afirst vector having repressible promoter that controls the expression ofan interfering RNA specific for a first gene product involved innicotine biosynthesis, a second vector having a constitutive promoterthat controls the expression of an interfering RNA specific for a secondgene product involved in nicotine biosynthesis and a third vector havingan inducible promoter that controls the expression of an interfering RNAspecific for a third gene product involved in nicotine biosynthesis.

In other embodiments provided herein, interfering RNAs can be producedsynthetically and introduced into a cell by methods known in the art.Synthetic interfering RNAs can include a variety of RNA molecules, whichinclude, but are not limited to, nucleic acids having at least oneregion of duplex RNA. The duplex RNA in such molecules can comprise, forexample, two antiparallel RNA strands that form a double-stranded RNAhaving flush ends, two antiparallel RNA strands that form adouble-stranded RNA having at least one end that forms a hairpinstructure, or two antiparallel RNA strands that form a double-strandedRNA, wherein both ends form a hairpin structure. In some embodiments,synthetic interfering RNAs comprise a plurality of RNA duplexes.

The regions of RNA duplex in synthetic interfering RNAs can range fromabout 10 bp in length to about 10,000 bp in length. In some embodiments,the duplex RNA ranges from about 15 bp in length to about 1500 bp inlength. In other embodiments, the duplex RNA ranges from about 20 bp inlength to about 1200 bp in length. In still other embodiments, theduplex RNA ranges from about 21 bp in length to about 23 bp in length.In a preferred embodiment, the duplex RNA has a length of 22 bps. Inpreferred embodiments, synthetic interfering RNAs are siRNAs. In anotherpreferred embodiment, the synthetic interfering RNA is an siRNA specificfor the coding sequence of a target mRNA encoding a polypeptide involvedin nicotine biosynthesis. In another preferred embodiment, the syntheticinterfering RNA is an siRNA specific for the coding sequence of a targetmRNA encoding a polypeptide involved in sterol biosynthesis.

Some embodiments provided herein relate to interfering nucleic acidsthat are not comprised entirely of RNA. Still other aspects relate tointerfering nucleic acids that do not comprise any RNA. Such interferingnucleic acids are synthetic interfering RNA analogs. These analogssubstantially mimic the specificity and activity of interfering RNA fromwhich they are modeled; however, they typically include additionalproperties which make their use desirable. For example, one or bothstrands of the interfering nucleic acid may contain one or morenonnatural nucleotide bases that improve the stability of the molecule,enhance that affinity of the molecule for the target mRNA and/or enhancecellular uptake of the molecule. Other modifications are alsocontemplated. For example, an interfering nucleic acid can include oneor more nucleic acid strands composed of naturally-occurringnucleobases, sugars and covalent internucleoside (backbone) linkages aswell as non-naturally-occurring nucleobases, sugars and covalentinternucleoside linkages.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or the 5′hydroxyl moiety of the sugar. In forming nucleic acids, the phosphategroups covalently link adjacent nucleosides to one another to form alinear polymeric compound. In turn the respective ends of this linearpolymeric structure can be further joined to form a circular structure.Within the nucleic acid structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Specific examples of interfering nucleic acids useful in certainembodiments of provided herein include one or more nucleic acid strandscontaining modified backbones or non-natural internucleoside linkages.As used herein, nucleic acids having modified backbones include thosethat retain a phosphorus atom in the backbone and those that do not havea phosphorus atom in the backbone.

In some embodiments, modified nucleic acid backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Certain nucleic acids having inverted polarity comprise a single 3′ to3′ linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

In some embodiments, modified nucleic acid backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

In other embodiments, the interfering nucleic acid can comprise one ormore mimetic regions, wherein both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. In such embodiments, the base units are maintained forhybridization with an appropriate nucleic acid target compound. One suchcompound, a mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone, in particular an aminoethylglycine backbone.The nucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. RepresentativeUnited States patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference in itsentirety. Further teaching of PNA compounds can be found in Nielsen etal., Science, 1991, 254, 1497-1500.

In still other embodiments provided herein, interfering nucleic acidsmay include nucleic acid strands having phosphorothioate backbonesand/or heteroatom backbones. Modified interfering nucleic acids may alsocontain one or more substituted sugar moieties. In some embodiments, theinterfering nucleic acids comprise one of the following at the 2′position: OH; F; O-, S-, or N-alkyl; S-, or N-alkenyl; O-, S- orN-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂ andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties.Another modification includes 2′-methoxyethoxy (2′ OCH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504).

An embodiment provided herein includes the use of Locked Nucleic Acids(LNAs) to generate interfering nucleic acids having enhanced affinityand specificity for the target polynucleotide. LNAs are nucleic acid inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety. The linkage ispreferably a methylene (—CH₂—)n group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in WO 98/39352 and WO 99/14226, the disclosures of which areincorporated herein by reference in their entireties.

Other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Interfering nucleic acids may also have sugar mimetics such ascyclobutyl moieties in place of the pentofuranosyl sugar.

The interfering nucleic acids contemplated herein may also includenucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine and other alkynyl derivativesof pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazi-n-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrimido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993, the disclosures of which are incorporated hereinby reference in their entireties. Certain of these nucleobases areparticularly useful for increasing the binding affinity of theinterfering nucleic acids described herein. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Another modification of the interfering nucleic acids described hereininvolves chemically linking to at least one of the nucleic acid strandsone or more moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the of the interfering nucleic acid.The interfering nucleic acids can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of nucleic acids, and groups that enhance thepharmacokinetic properties of such molecules. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improveinterfering nucleic acid uptake, enhance its resistance to degradation,and/or strengthen sequence-specific hybridization with target molecules.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve the uptake, distribution,metabolism or excretion of the interfering nucleic acid. Conjugatemoieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.,1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al.,Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., dihexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

As described above, it is not necessary for all positions in a givencompound to be uniformly modified, and in fact, more than one of theaforementioned modifications may be incorporated in a single compound oreven at a single nucleoside within a nucleic acid. The methods describedherein also contemplate the use of interfering nucleic acids which arechimeric compounds. “Chimeric” interfering nucleic acid compounds or“chimeras,” as used herein, are interfering nucleic acid compounds,which contain two or more chemically distinct regions, each made up ofat least one monomer unit, i.e., a nucleotide in the case of a nucleicacid compound. These interfering nucleic acids typically contain atleast one region wherein the nucleic acid is modified so as to conferupon the interfering nucleic acid increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid. An additional region of thenucleic acid may serve as a substrate for enzymes capable of cleavingRNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby contributes further to the inhibition of gene expression by theinterfering nucleic acid.

The above-described interfering nucleic acids may be conveniently androutinely made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare nucleic acids such as the phosphorothioates and alkylatedderivatives.

The interfering nucleic acid compounds for use with the methodsdescribed herein encompass any pharmaceutically acceptable salts,esters, or salts of such esters, or any other compound.

Although terms, such as interfering RNA, dsRNA and siRNA, are usedthroughout the remainder of the specification, it will be appreciatedthat in the context of synthetically produced interfering nucleic acids,that such terms are meant to include interfering nucleic acids of alltypes, including those which incorporate modifications, such as thosedescribed above.

Some embodiments provided herein relate to methods of reducing oreliminating the expression of one or more target genes involved innicotine, nornicotine, and/or sterol biosynthesis. Target genes that areinvolved in nicotine, nornicotine, and/or sterol biosynthesis areexpressed through the transcription a first gene product, the targetmRNA, which is then translated to produce a second gene product, thetarget polypeptide. Thus, reduction or elimination of the expression ofone or more target genes results in the reduction or elimination of oneor more target mRNAs and/or target polypeptides. Target polypeptidesinvolved in nicotine and nornicotine biosynthesis include, for example,putrescene N-methyltransferase, N-methylputrescene oxidase, ornithinedecarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase,phosphoribosylanthranilate isomerase, and quinolate phosphoribosyltransferase (QPTase). In a preferred embodiment, the expression of theQPTase, PMTase, and A622 product is inhibited. Target polypeptidesinvolved in sterol biosynthesis include, for example, HMG-CoA reductase,14alpha demethylase, squalene synthase, SMT2, SMT1, C14 sterolreductase, A8-A7-isomerase, and C4-demethylase.

Reduction of the expression of one or more target genes and/or targetgene products that are involved in nicotine, nornicotine, and/or sterolbiosynthesis leads to a reduction in the amount of nicotine, sterols,and TSNAs produced in tobacco and PAHs upon pyrolysis of the tobacco. Incertain embodiments, the expression of one or more target gene productsinvolved in nicotine, nornicotine, and/or sterol biosynthesis iseliminated. Elimination of such target gene products can result in theelimination of nicotine, nornicotine, and/or sterol biosynthesis,thereby reducing the amount of nicotine, nornicotine, and/or sterolpresent in tobacco to levels below the detection limit of methodscommonly used. Reduction of the amount of nicotine and nornicotinepresent in tobacco can lead to a reduction in the amount of TSNAsproduced in the tobacco. In some embodiments, the amount of TSNA presentin tobacco is reduced to levels below the detection limit of methodscommonly used to detect TSNAs. Similarly, the reduction in the amount ofsterol present in tobacco can lead to a reduction in the amount of PAHgenerated from the tobacco upon pyrolysis. In some embodiments, theamount of PAH present in tobacco is reduced to levels below thedetection limit of methods commonly used to detect PAH.

The reduction in or elimination of the expression of target genes ortarget gene products involved in nicotine, nornicotine, and/or sterolbiosynthesis is achieved by providing an interfering RNA specific to oneor more such target genes to a tobacco cell, thereby producing agenetically modified tobacco cell. The interfering RNA can be providedas a synthetic double-stranded RNA, or alternatively, as a nucleic acidconstruct capable of encoding the interfering RNA. Syntheticdouble-stranded interfering RNAs are taken up by the cell directlywhereas interfering RNAs encoded by a nucleic acid construct areexpressed from the construct subsequent to the entry of the constructinside the cell. The reduction in or elimination of the expression ofthe target genes and/or the target gene products is mediated by thepresence of the interfering RNA inside the cell.

In general, the interfering RNAs that are produced inside the cell,whether expressed from a nucleic acid construct or provided as syntheticdouble-stranded RNA molecules, include an RNA duplex having a first andsecond strand. At least a portion the first strand of the duplex issubstantially similar or identical to at least a portion of a targetmRNA or a target gene involved in nicotine biosynthesis.Correspondingly, at least a portion of the second strand of the duplexis complementary or substantially complementary to the first strand, andthus, at least a portion of the second strand is complementary orsubstantially complementary to at least a portion of the mRNA encoded bythe target gene. In some embodiments provided herein, the interferingRNA can comprise a first strand that is substantially similar oridentical to the entire coding sequence of the target gene or targetmRNA involved in nicotine biosynthesis and a second strand complementaryor substantially complementary to the first strand. In some embodimentsprovided herein, the interfering RNA can comprise a first strand that issubstantially similar or identical to the entire coding sequence of thetarget gene or target mRNA involved in sterol biosynthesis and a secondstrand complementary or substantially complementary to the first strand.

The reduction in or elimination of the expression of genes and/or geneproducts involved in nicotine, nornicotine, and/or sterol biosynthesiscan be characterized by comparing the amount of nicotine, nornicotine,and/or sterol produced in genetically modified cells, with the amount ofnicotine, nornicotine, and/or sterol produced in cells that have notbeen genetically modified. Alternatively, such reduction in orelimination of gene expression can be characterized by geneticallyanalyzing plant cells so as to determine the level of mRNA present inthe genetically modified plant cell as compared to a non-modified plantcell. Depending on the assay, quantitation of the amount of geneexpression allows one to determine a degree of reduction in geneexpression, which can be greater than 10%, 33%, 50%, 90%, 95% or 99% ascompared to an untreated cell. As with nicotine and nornicotine, thereduction in or elimination of TSNA production in tobacco can becharacterized by comparing the amount of TSNAs produced in geneticallymodified cells, with the amount of TSNAs produced in cells that have notbeen genetically modified. The section below provides more descriptionof the transgenic plants and cells provided herein.

b) Transgenics

Transgenic Plant Cells and Plants

Embodiments provided herein concern transgenic plant cells comprisingone or more interfering RNAs that are capable of reducing or eliminatingthe expression of one or more target genes and/or target gene productsinvolved in nicotine, nornicotine, and/or sterol biosynthesis. Asdescribed above, an appropriate interfering RNA comprises a duplex RNAthat comprises a first strand that is substantially similar or identicalto at least a portion of a target gene or target mRNA, which encodes agene product involved in nicotine, nornicotine, and/or sterolbiosynthesis. The RNA duplex also comprises a second strand that iscomplementary or substantially complementary to the first strand.

The interfering RNA or nucleic acid construct comprising the interferingRNA can be introduced into the plant cell in any suitable manner. Plantcells possessing stable interfering RNA activity, for example, by havinga nucleic acid construct stably integrated into a chromosome, can beused to regenerate whole plants using methods known in the art. As such,some embodiments provided herein relate to plants, such as tobaccoplants, transformed with one or more nucleic acid constructs and/orvectors which encode at least one interfering RNA that is capable ofreducing or eliminating the expression of a gene product involved innicotine biosynthesis. Transgenic tobacco cells and the plants describedherein are characterized in that they have a reduced amount of nicotine,nornicotine, sterol and/or TSNA and/or generate a reduced amount of PAHsupon pyrolysis, as compared to unmodified or control tobacco cells andplants.

The tobacco plants described herein are suitable for conventionalgrowing and harvesting techniques (e.g. topping or no topping, baggingthe flowers or not bagging the flowers, cultivation in manure rich soilor without manure) and the harvested leaves and stems are suitable foruse in any traditional tobacco product including, but not limited to,pipe, cigar and cigarette tobacco and chewing tobacco in any formincluding leaf tobacco, shredded tobacco or cut tobacco. It is alsocontemplated that the low nicotine and/or TSNA tobacco described hereincan be processed and blended with conventional tobacco so as to create awide-range of tobacco products with varying amounts of nicotine and/orTSNAs. These blended tobacco products can be used in tobacco productcessation programs so as to slowly move a consumer from a high nicotineand/or sterol product to a low nicotine and/or sterol product. Someembodiments provided herein comprise a tobacco use cessation kit,comprising two or more tobacco products with different levels ofnicotine. For example, a smoker can begin the program smoking blendedcigarettes having or delivering 0.6 mg of nicotine, gradually move tosmoking cigarettes having or delivering 0.3 mg of nicotine, followed bycigarettes having or delivering less than 0.1 mg nicotine until theconsumer decides to quit smoking altogether. Accordingly, the blendedcigarettes described herein provide the basis for an approach to reducethe exposure of a tobacco consumer to a tobacco related disease in astep-wise fashion. The components of the tobacco use cessation kitdescribed herein may include other tobacco products, including but notlimited to, smoking materials (e.g., cigarettes, cigars, pipe tobacco),snuff, chewing tobacco, gum, and lozenges.

Gene silencing has been employed in several laboratories to createtransgenic plants characterized by lower than normal amounts of specificgene products. As used herein, “exogenous” or “heterologous” nucleicacids, including DNAs and/or RNAs, refer to nucleic acids that have beenintroduced into a cell (or the cell's ancestor) through the efforts ofhumans. The nucleic acid constructs that are used with the transgenicplants and the methods for producing the transgenic plants describedherein encode one or more interfering RNA constructs comprisingregulatory sequences, which include, but are not limited to, atranscription initiation sequence (“promoter”) operable in the plantbeing transformed, and a polyadenylation/transcription terminationsequence. Typically, the promoter is located upstream of the 5′-end ofthe nucleotide sequence to be expressed. The transcription terminationsequence is generally located just downstream of the 3′-end of thenucleotide sequence to be transcribed.

In some preferred embodiments, the nucleic acid encoding the exogenousinterfering RNA, which is transformed into a tobacco cell, comprises afirst RNA strand that is identical to the an endogenous coding sequenceof a gene encoding a gene product involved in nicotine biosynthesis.However, minor variations between the exogenous and endogenous sequencescan be tolerated. It is preferred, but not necessarily required, thatthe exogenously-produced interfering RNA sequence, which issubstantially similar to the endogenous gene coding sequence, be ofsufficient similarity to the endogenous gene coding sequence, such thatthe complementary interfering RNA strand is capable of binding to theendogenous sequence in the cell to be regulated under stringentconditions as described below.

In some embodiments, the heterologous sequence utilized in the methodsprovided herein may be selected so as to produce an interfering RNAproduct comprising a first strand that is substantially similar oridentical to the entire QTPase mRNA sequence, or to a portion thereof,and a second strand that is complementary to the entire QPTase mRNAsequence, or to a portion thereof. The interfering RNA may becomplementary to any contiguous sequence of the natural messenger RNA.For example, it may be complementary to the endogenous mRNA sequenceproximal to the 5′-terminus or capping site, downstream from the cappingsite, between the capping site and the initiation codon and may coverall or only a portion of the non-coding region, may bridge thenon-coding and coding region, be complementary to all or part of thecoding region, complementary to the C-terminus of the coding region, orcomplementary to the 3′-untranslated region of the mRNA.

As used herein, the term “gene” refers to a DNA sequence thatincorporates (1) upstream (5′) regulatory signals including thepromoter, (2) a coding region specifying the product, protein or RNA ofthe gene, (3) downstream regions including transcription termination andpolyadenylation signals and (4) associated sequences required forefficient and specific expression. The DNA sequence provided herein mayconsist essentially of the sequence provided herein, or equivalentnucleotide sequences representing alleles or polymorphic variants ofthese genes, or coding regions thereof. Use of the phrase “substantialsequence similarity” or “substantially similar” in the presentspecification and claims means that DNA, RNA or amino acid sequenceswhich have slight and non-consequential sequence variations from theactual sequences disclosed and claimed herein are considered to beequivalent to the sequences provided herein. In this regard, “slight andnon-consequential sequence variations” mean that “similar” sequences(i.e., the sequences that have substantial sequence similarity with theDNA, RNA or proteins disclosed and claimed herein) will be functionallyequivalent to the sequences disclosed and claimed in the presentinvention. Functionally equivalent sequences will function insubstantially the same manner to produce substantially the samecompositions as the nucleic acid and amino acid compositions disclosedand claimed herein.

As used herein, a “native nucleotide sequence” or “natural nucleotidesequence” means a nucleotide sequence that can be isolated fromnon-transgenic cells or tissue. Native nucleotide sequences are thosewhich have not been artificially altered, such as by site-directedmutagenesis. Once native nucleotide sequences are identified, nucleicacid molecules having native nucleotide sequences may be chemicallysynthesized or produced using recombinant nucleic acid procedures as areknown in the art. As used herein, a “native plant nucleotide sequence”is that which can be isolated from non-transgenic plant cells or tissue.As used herein, a “native tobacco nucleotide sequence” is that which canbe isolated from non-transgenic tobacco cells or tissue. Use of thephrase “isolated” or “substantially pure” in the present specificationand claims as a modifier of nucleic acids, polypeptides or proteinsmeans that the nucleic acids, polypeptides or proteins so designatedhave been separated from their in vivo cellular environments through theefforts of human beings.

The nucleotide sequences provided herein, such as interfering RNAs ornucleic acids encoding interfering RNAs, can be transformed into avariety of host cells. As used herein, “transformation” refers to theintroduction of exogenous nucleic acid into cells so as to producetransgenic cells stably transformed with the exogenous nucleic acid. Avariety of suitable host cells, having desirable growth and handlingproperties, are readily available in the art.

Standard techniques, such as restriction mapping, Southern blothybridization, polymerase chain reaction (PCR) and/or nucleotidesequence analysis can be employed to identify clones expressing thedesired interfering RNA construct. Following the introduction andverification of the desired interfering RNA or nucleic acid constructencoding the desired interfering RNA, whole plants can be regeneratedfrom successfully transformed cells using conventional techniques.

Nucleic acid constructs, or “transcription cassettes,” encoding theinterfering RNAs that are used to produce the transgenic cells andplants provided herein include, 5′ to 3′ in the direction oftranscription, a promoter as described herein, a nucleotide sequence asdescribed herein operatively associated with the promoter, and,optionally, a termination sequence including stop signal for RNApolymerase and a polyadenylation signal. All of these regulatory regionsshould be capable of operating in the cells of the tissue to betransformed. Any suitable termination signal may be employed in carryingout the present invention, examples thereof including, but not limitedto, the nopaline synthase (nos) terminator, the octapine synthase (ocs)terminator, the CaMV terminator or native termination signals, derivedfrom the same gene as the transcriptional initiation region or derivedfrom a different gene. (See, e.g., Rezian et al. (1988) supra, andRodermel et al. (1988), supra).

The term “operatively associated,” as used herein, refers to nucleotidesequences on a single nucleic acid molecule that are associated so thatthe function of one sequence is affected by the other. Thus, a promoteris operatively associated with a nucleotide sequence when it is capableof affecting the transcription of that sequence (i.e., the nucleic acidis under the transcriptional control of the promoter). The promoter issaid to be “upstream” from the transcribed nucleotide sequence, which isin turn said to be “downstream” from the promoter.

In some embodiments, the transcription cassette may be provided in a DNAconstruct that also has at least one replication system. Forconvenience, it is common to have a replication system functional inEscherichia coli, such as ColE1, pSC101, pACYC184, or the like. In thismanner, at each stage after each manipulation, the resulting constructmay be cloned, sequenced, and the correctness of the manipulationdetermined. In addition, or in place of the E. coli replication system,a broad host range replication system may be employed, such as thereplication systems of the P-1 incompatibility plasmids, e.g., pRK290.In addition to the replication system, there will frequently be at leastone marker present, which may be useful in one or more hosts, ordifferent markers for individual hosts. That is, one marker may beemployed for selection in a prokaryotic host, while another marker maybe employed for selection in a eukaryotic host, particularly the planthost. The markers may be protection against a biocide (such asantibiotics, toxins, heavy metals or the like), provide complementationby imparting prototrophy to an auxotrophic host and/or provide a visiblephenotype through the production of a novel compound in the plant.

The various fragments comprising the various constructs, transcriptioncassettes, markers and the like may be introduced consecutively byrestriction enzyme cleavage of an appropriate replication system andinsertion of the particular construct or fragment into the availablesite. After ligation and cloning, the DNA construct may be isolated forfurther manipulation. All of these techniques are amply exemplified inthe literature as demonstrated by J. Sambrook et al., Molecular Cloning,A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).

Vectors that may be used to transform plant tissue with nucleic acidconstructs provided herein include Agrobacterium and Transbacter vectorsand ballistic vectors, as well as vectors suitable for DNA-mediatedtransformation. In this particular embodiment, the promoter is a regionof a DNA sequence that incorporates the necessary signals for theefficient expression of the coding sequence. This region may includesequences to which an RNA polymerase binds, but is not limited to suchsequences, and may include sequences to which other regulatory proteinsbind along with sequences involved in the control of proteintranslation. Such regions may also include coding sequences.

Promoters employed in carrying out the invention may be constitutivelyactive promoters. Numerous constitutively active promoters that areoperable in plants are available. A preferred example is the CauliflowerMosaic Virus (CaMV) 35S promoter, which is expressed constitutively inmost plant tissues. As an alternative, the promoter may be aroot-specific promoter or root cortex specific promoter, as explained ingreater detail below.

Nucleic acid sequences have been expressed in transgenic tobacco plantsutilizing the Cauliflower Mosaic Virus (CaMV) 35S promoter. (See, e.g.,Cornelissen et al., “Both RNA Level and Translation Efficiency areReduced by Anti-Sense RNA in Transgenic Tobacco”, Nucleic Acids Res. 17,pp. 833-43 (1989); Rezaian et al., “Anti-Sense RNAs of Cucumber MosaicVirus in Transgenic Plants Assessed for Control of the Virus”, PlantMolecular Biology 11, pp. 463-71 (1988); Rodermel et al.,“Nuclear-Organelle Interactions: Nuclear Antisense Gene InhibitsRibulose Bisphosphate Carboxylase Enzyme Levels in Transformed TobaccoPlants”, Cell 55, pp. 673-81 (1988); Smith et al., “Antisense RNAInhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes”,Nature 334, pp. 724-26 (1988); Van der Krol et al., “An Anti-SenseChalcone Synthase Gene in Transgenic Plants Inhibits FlowerPigmentation”, Nature 333, pp. 866-69 (1988)).

Use of the CaMV 35S promoter for expression of interfering RNAs in thetransformed tobacco cells and plants provided herein is preferred. Useof the CaMV promoter for expression of other recombinant genes intobacco roots has been well described (Lam et al., “Site-SpecificMutations Alter In Vitro Factor Binding and Change Promoter ExpressionPattern in Transgenic Plants”, Proc. Nat. Acad Sci. USA 86, pp. 7890-94(1989); Poulsen et al. “Dissection of 5′ Upstream Sequences forSelective Expression of the Nicotiana plumbaginifolia rbcS-8B Gene”,Mol. Gen. Genet. 214, pp. 16-23 (1988)). Other promoters that are activeonly in root tissues (root specific promoters) are also particularlysuited to the methods provided herein. See, e.g., U.S. Pat. No.5,459,252 to Conkling et al.; Yamamoto et al., The Plant Cell, 3:371(1991). The TobRD2 root-cortex specific promoter may also be utilized.All patents cited herein are intended to be incorporated herein byreference in their entirety.

The recombinant interfering nucleic acid molecules and vectors used toproduce the transformed tobacco cells and plants provided herein mayfurther comprise a dominant selectable marker gene. Suitable dominantselectable markers for use in tobacco include, inter alia, antibioticresistance genes encoding neomycin phosphotransferase (NPTII) andhygromycin phosphotransferase (HPT). Preferred selectable markersinclude the norflurazone resistance genes described in this disclosure.Other well-known selectable markers that are suitable for use in tobaccoinclude a mutant dihydrofolate reductase gene that encodesmethotrexate-resistant dihydrofolate reductase. DNA vectors containingsuitable antibiotic resistance genes, and the corresponding antibiotics,are commercially available.

Transformed tobacco cells are selected out of the surrounding populationof non-transformed cells by placing the mixed population of cells into aculture medium containing an appropriate concentration of the antibiotic(or other compound normally toxic to tobacco cells) against which thechosen dominant selectable marker gene product confers resistance. Thus,only those tobacco cells that have been transformed will survive andmultiply. Additionally, the positive selection techniques described byJefferson (e.g., WO 00055333; WO 09913085; U.S. Pat. Nos. 5,599,670;5,432,081; and 5,268,463, hereby expressly incorporated by reference intheir entireties) can be used.

Methods of making recombinant plants provided herein, in general,involve first providing a plant cell capable of regeneration (the plantcell typically residing in a tissue capable of regeneration). The plantcell is then transformed with an interfering RNA or a nucleic acidconstruct encoding an interfering RNA comprising a transcriptioncassette provided herein (as described above) and a recombinant plant isregenerated from the transformed plant cell. As explained below, thetransforming step is carried out by techniques as are known in the art,including but not limited to bombarding the plant cell withmicroparticles carrying the transcription cassette, infecting the cellwith an Agrobacterium tumefaciens containing a Ti plasmid carrying thetranscription cassette or any other technique suitable for theproduction of a transgenic plant.

Numerous Agrobacterium vector systems useful in carrying out the presentinvention are known. For example, U.S. Pat. No. 4,459,355 discloses amethod for transforming susceptible plants, including dicots, with anAgrobacterium strain containing the Ti plasmid. The transformation ofwoody plants with an Agrobacterium vector is disclosed in U.S. Pat. No.4,795,855. Further, U.S. Pat. No. 4,940,838 to Schilperoort et al.discloses a binary Agrobacterium vector (i.e., one in which theAgrobacterium contains one plasmid having the vir region of a Ti plasmidbut no T region, and a second plasmid having a T region but no virregion) useful in carrying out the present invention, all references arehereby expressly incorporated by reference in their entireties.

Microparticles suitable for the ballistic transformation of a plantcell, carrying a nucleic acid construct provided herein, are also usefulfor making the transformed plants described herein. The microparticle ispropelled into a plant cell to produce a transformed plant cell and aplant is regenerated from the transformed plant cell. Any suitableballistic cell transformation methodology and apparatus can be used inpracticing the present invention. Exemplary apparatus and procedures aredisclosed in Sanford and Wolf, U.S. Pat. No. 4,945,050, and in Christouet al., U.S. Pat. No. 5,015,580. When using ballistic transformationprocedures, the transcription cassette may be incorporated into aplasmid capable of replicating in or integrating into the cell to betransformed. Examples of microparticles suitable for use in such systemsinclude 1 to 5 μm gold spheres. The nucleic acid construct may bedeposited on the microparticle by any suitable technique, such as byprecipitation.

Plant species may be transformed with the interfering RNA or nucleicacid construct encoding an interfering RNA provided herein by thenucleic acid-mediated transformation of plant cell protoplasts. Plantsmay be subsequently regenerated from the transformed protoplasts inaccordance with procedures well known in the art. Fusion of tobaccoprotoplasts with nucleic acid-containing liposomes or with nucleic acidconstructs via electroporation is known in the art. (Shillito et al.,“Direct Gene Transfer to Protoplasts of Dicotyledonous andMonocotyledonous Plants by a Number of Methods, IncludingElectroporation”, Methods in Enzymology 153, pp. 313-36 (1987)).

These inhibition constructs or RNAi constructs can be transferred toplant cells by any known method in the art. Preferably,Agrobacterium-mediated or Biolistic-mediated transformation are used,according to well-established protocols. It is also contemplated thatTransbacter-mediated transformation can be used, as described below.(See Broothaerts et al., Nature 433, 629 (2005), herein expresslyincorporated by reference in its entirety).

By this approach, first bacteria are prepared as follows. YM plusantibiotic plates (see below) are streaked with bacteria and the platesare incubated for 2-3 days at 28° C. Transformation is accomplished bymeasuring about 20 mL Minimal A medium for each bacterial strain.Scrapping or washing the Scrape or wash bacteria from plate with sterileloop and then suspending said bacteria in 20 mL of Minimal A medium. Thecell density is adjusted to an OD600 0.9-1.0.

Next, the first healthy fully expanded leaves from 4-5 week old tissueculture grown tobacco plants are cut into 0.5 cm squares (or can use acork borer, which is about 1.0 cm diameter) in deep petri dish, understerile RMOP liquid medium. The tissue pieces are stored in RMOP in adeep petri dish. The leaf pieces (about 20 per transformation) are thentransferred to a deep petri dish containing bacterial suspension. Toensure that the bacteria have contacted a cut edge of the leaf, thesuspension with leaf cutting is swirled and is left standing for 5minutes. The leaf pieces are then removed from the suspension andblotted dry on filter paper or on the edge of the container. The leafpieces are then placed with adaxial side (upper leaf surface) on solidRMOP at about 10 pieces per plate.

The plates are then incubated in the dark at 28° C. for: 2-3 days, if A.tumefaciens is used, 5 days if S. mehlotiis used, 5 days M. loti isused, and 5-11 days if Rhizobium sp. NGR234 is used. Over the next week,selection is performed. For the purposes of this example, hygromycinselection is performed. Accordingly, the leaf pieces are transferredonto solid RMOP-TCH, with abaxial surface (lower surface of leaf) incontact with media.

The plates are incubated for 2-3 weeks in the light at 28° C., with 16hours daylight per day. Subculture occurs every 2 weeks.

Plantlet formation is accomplished as follows. Once shoots appear, theplantlet is transferred to MST-TCH pots. The plantlets are grown with 16hours daylight for 1-2 weeks. Once roots form the plants appear, theplants can be transferred to soil in the greenhouse.

Media and Solutions for Tobacco Transformation:

YM Media (1 L)

Mannitol 10 g Yeast extract 0.4 g K2HPO4 (10% w/v stock) 1 ml KH2PO4(10% w/v stock) 4 ml NaCl (10% w/v stock) 1 ml MgSO4•7H2O (10% w/vstock) 2 ml pH 6.8 Agar 15 g/L Autoclave *When ready to pour addantibiotic selection if required

Keep poured plates for 2 days at room temperature to visualize anycontamination, then store at 4° C.

RMOP+RMOP-TCH Media

(Svab, Z., et al., 1975. Transgenic tobacco plants by cocultivation ofleaf disks with pPZP Agrobacterium binary vectors. In “Methods in PlantMolecular Biology-A Laboratory Manual”, P. Maliga, D. Klessig, A.Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press:55-77), herein expressly incorporated by reference in its entirety).

1 L Final Conc.

Sucrose 30 g   (3%) Myo-inositol 100 mg (0.1%) MS Macro 10x 100 mL (1x)MS Micro 1000x 1 mL (1x) Fe2EDTA Iron 100x 10 mL (1x) Thiamine-HCl (10mg/mL stock) 100 μL (1 mg) NAA (1 mg/mL stock) 100 μL 0.1 mg) BAP (1mg/mL stock) 1 mL (1 mg) pH 5.8 Phytagel 2.5 g/L for solid autoclave*for RMOP-TCH, when ready to pour add: Timentin (200 mg/mL stock) 1 mL,Claforan (250 mg/mL stock) 1 mL, and Hygromycin (50 mg/mL stock) 1 mLBAP (1 mg/ml) (6-Benzylaminopurine)

Add 1N KOH drop wise to 100 mg BAP until dissolved. Make up to 100 Mlwith Milli-Q H2O and store at 4° C.

NAA (1 mg/ml) (Naphthalene Acetic Acid)

Dissolve 100 mg NAA in 1 mL absolute ethanol. Add 3 mL 1N KOH. Make upto 80 mL with Milli-Q H2O. Adjust pH to 6.0 with 1N HCl, make up to 100mL with Milli-Q H2O, and store at 4° C.

Cefotaxamine (250 mg/ml)

Add 8 ml sterile Milli-Q H2O to 2 g Claforan and store at 4° C. in dark

Timentin (200 mg/ml)

Add 15 ml sterile Milli-Q H2O to 3 g Timentin and store at 4° C.

MST+MST-TCH Media

(Svab, Z., et al., 1975. Transgenic tobacco plants by cocultivation ofleaf disks with pPZP Agrobacterium binary vectors. In “Methods in PlantMolecular Biology-A Laboratory Manual”, P. Maliga, D. Klessig, A.,Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press:55-77), herein expressly incorporated by reference in its entirety).

1 L Final Concentration

Sucrose 30 g (3%) MS Macro 10x 100 mL (1x) MS Micro 1000x 1 mL (1x)Fe2EDTA Iron 100x 10 mL (1x) pH 5.8 Phytagel 2.5 g/L Autoclave ForMST-TCH, when ready to pour add: Timentin (200 mg/mL stock) (1 mL)Cefotaxamine (250 mg/mL stock) (1 mL) Hygromycin (50 mg/mL stock) (1 mL)MS Macro 10× ((Murashige and Skoog., Phys. Plant. 15: 473-497 (1962),herein expressly incorporated by reference in its entirety)).Final Concentration

10x (g/L) KNO3 19.0 NH4 N03 16.5 CaCl2•2H2O 4.4 MgS04•7H2O 3.7 KH2PO41.7 Store 4° C. Substituting chemicals: CaCl2 3.3 g/L MgS04 1.8 g/LMS Micro 1000× (Murashige and Skoog., Phys. Plant. 15: 473-497 (1962),herein expressly incorporated by reference in its entirety).Final Concentration

1000x (g/L) MnS04•4H20 22.3 ZnS04•7H20 8.6 H3BO3 6.2 KI 0.83Na2MoO4•2H2O 0.25 CuSO4•5H2O 25 mg CoCl2•6H2O 25 mg Store 4° C.Substituting chemicals: MnS04•H20 16.9/LFeSO4EDTA Iron 100×

(g/1 L) FeS04•7H20 2.78 Na2EDTA 3.72 Store 4° C. in dark bottle

Once the transformed cells are selected, by any of the approachesdescribed above, they are induced to regenerate intact tobacco plantsthrough application of tobacco cell and tissue culture techniques thatare well known in the art. The method of plant regeneration is chosen soas to be compatible with the method of transformation. The stablepresence of an interfering RNA or a nucleic acid encoding an interferingRNA in transgenic tobacco plants can be verified by Mendelianinheritance of the interfering RNA or a nucleic acid encoding aninterfering RNA sequence, as revealed by standard methods of nucleicacid analysis applied to progeny resulting from controlled crosses.After regeneration of transgenic tobacco plants from transformed cells,the introduced nucleic acid sequence can be readily transferred to othertobacco varieties through conventional plant breeding practices andwithout undue experimentation.

For example, to analyze the segregation of the transgene, regeneratedtransformed plants (TO) may be grown to maturity, tested for nicotineand/or TSNA levels, and selfed to produce T₁ plants. A percentage of T₁plants carrying the transgene are homozygous for the transgene. Toidentify homozygous T₁ plants, transgenic T₁ plants are grown tomaturity and selfed. Homozygous T₁ plants will produce T₂ progeny whereeach progeny plant carries the transgene; progeny of heterozygous T₁,plants will segregate 3:1.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a nucleic acidembodiment provided herein. Preferred plants for introduction of anucleic acid embodiment, described herein, include Nicotiana. Preferredvarieties of Nicotiana for introduction of a nucleic acid embodiment asdescribed herein include the Nicotiana tabacum varieties provided inTable 1.

TABLE 1 Burley Dark Flu One Newest Varieties Varieties Cured OtherVirginia Hybrid Sucker Varieties Oriental KT 200 BLACK CU BROWN NBH LCMAMMOTH K 149 748 LEAF 98 OS400 GL 350 D174 LIZARD MS KT 204 GL TAIL 21× KY KY LC DF 485 K 326 737 ORNOCO 10 160 Izmir LIZARD TAIL MS OX TURTLE14 × KY KY DF 911 K 346 207 FOOT L8 PVH KY 10 DT 508 K 394 03 M and N TN97 PVH KY 14 DT 518 K 730 09 SHIREY KT 200 Coker PVH WALKER KY 17 DT 592371 Gold 2040 BROADLEAF GREEN KY 907 WOOD CU 748 RG 17 KY 907 IMPROVEDLC MADOLE GL 737 RG 81 KY 908 KT-D4 LC GL 939 RGH 4 RGH KY 908 KY 160 GL973 51 RS KY 910 KY 171 K 358 1410 MS Burley 21 × KY Speight 10 KY 171 K399 168 MS KY14 × LITTLE Speight L8 CRITTENDEN NC 102 179 LITTLE SpeightN 126 WOOD NC 291 190 NARROW LEAF Speight N 777 MADOLE NC 297 196NEWTON'S Speight N 88 VH MADOLE NC 55 200A Speight NBH 98 NL MADOLE NC606 210 Speight TN 86 TN D94 NC 71 218 TN 86 Speight LC TN D950 NC 72220 TN 90 TR MADOLE NC 810 Speight H-20 TN 90 Speight LC VA 309 RGH 4H-6 TN 97 Speight LC VA 312 RGH 51 NF-3 VA VA 509 VA 355 119 NC 37 LA21VA 359 NF OX 414 NF Sp. G- 172

The term “organogenesis,” as used herein, means a process by whichshoots and roots are developed sequentially from meristematic centers;the term “embryogenesis,” as used herein, means a process by whichshoots and roots develop together in a concerted fashion (notsequentially), whether from somatic cells or gametes. The particulartissue chosen will vary depending on the clonal propagation systemsavailable for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, callus tissue, existing meristematictissue (e.g., apical meristems, axillary buds, and root meristems) andinduced meristem tissue (e.g., cotyledon meristem and hypocotylmeristem).

Plants provided herein may take a variety of forms. The plants may bechimeras of transformed cells and non-transformed cells; the plants maybe clonal transformants (e.g., all cells transformed to contain thetranscription cassette); the plants may comprise grafts of transformedand untransformed tissues (e.g., a transformed root stock grafted to anuntransformed scion in citrus species). The transformed plants may bepropagated by a variety of means, such as by clonal propagation orclassical breeding techniques. For example, first generation (or T₁)transformed plants may be selfed to give homozygous second generation(or T₂) transformed plants and the T₂ plants further propagated throughclassical breeding techniques. A dominant selectable marker (such asnptII) can be associated with the transcription cassette to assist inbreeding.

As used herein, a crop comprises a plurality of plants provided herein,and of the same genus, planted together in an agricultural field. By“agricultural field” is meant a common plot of soil or a greenhouse.Thus, the present invention provides a method of producing a crop ofplants having reduced amounts of nicotine, nornicotine, and/or sterol,as compared to a similar crop of non-transformed plants of the samespecies and variety.

The modified tobacco plants described herein are suitable forconventional growing and harvesting techniques (e.g. topping or notopping, bagging the flowers or not bagging the flowers, cultivation inmanure rich soil or without manure). The harvested tobacco leaves andstems are suitable for conventional methods of processing such as curingand blending. The modified tobacco is suitable for use in anytraditional tobacco product including, but not limited to, pipe, cigarand cigarette tobacco, and chewing tobacco in any form including leaftobacco, shredded tobacco, or cut tobacco.

Some embodiments concern the production and identification of particularlines of a transgenic Burley variety (Vector 21-41), which have very lowlevels of nicotine and TSNAs. The constructs used to create theseparticular lines of transgenic Burley tobacco are provided in Conklinget al., WO98/56923; U.S. Pat. Nos. 6,586,661; 6,423,520; and U.S. patentapplication Ser. Nos. 09/963,340; 10/356,076; 09/941,042; 10/363,069;10/729,121; 10/943,346, all of which are hereby expressly incorporatedby reference in their entireties. After the creation and analysis ofnearly 2,000 lines of transgenic Burley tobacco, these particular linesof reduced nicotine and TSNA transgenic tobacco were identified. Tobaccoharvested from these lines were incorporated into tobacco products(Quest 1®, Quest 2®, and Quest 3®) and were analyzed for their abilityto reduce the potential to contribute to a tobacco-related disease, asdescribed in the sections above. It was found that tobacco productscomprising these lines of transgenic Burley tobacco, had a reducedpotential to contribute to a tobacco-related disease (i.e., that thesetobacco products are reduced risk tobacco products).

3. Exemplary Constructs

Several embodiments concern isolated nucleic acids that comprise,consist, or consist essentially of the nucleic acids described in thesequence listing (SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49or 50) and fragments thereof at least 30 consecutive nucleotides inlength. That is, embodiments provided herein include an isolated nucleicacid comprising, consisting of, consisting essentially of, any one ormore of the sequences of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49 or 50, or a fragment thereof (e.g., a fragment that is at least,less than or equal to or greater than 30, 40, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380,400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660,680, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100,4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300,5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500,6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700,7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900,or 9000 consecutive nucleotides of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50.

In preferred embodiments, the target gene or target mRNA encodes QTPase,PMTase, or the A622 gene product. In preferred embodiments, aninterfering RNA comprises, consists, or consists essentially of an RNAstrand that is complementary to each least a portion (e.g., less than,greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500,750, or 1000 consecutive nucleotides) of SEQ ID NOS: 2, 3, 4, 5, 39 or40, and inhibits the production of QTPase, PMTase, A622, nicotine,nornicotine, NNN, NNK, NAT, or NAB in a tobacco. In related embodiments,the interfering RNA comprises, consists, or consists essentially of anRNA strand that is complementary to each least a portion (e.g., lessthan, greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150,250, 500, 750, or 1000 consecutive nucleotides) of SEQ ID NO: 5, andinhibits production of nornicotine but not nicotine in a tobacco. Instill more embodiments, the interfering RNA comprises, consists, orconsists essentially of an RNA strand that is complementary to eachleast a portion (e.g., less than, greater than or equal to 30, 35, 40,45, 50, 60, 75, 100, 150, 250, 500, 750, or 1000 consecutivenucleotides) of SEQ ID NO: 6, 7, 8, or 9, and inhibits production of atleast one sterol (e.g., squalene synthase, HMG-CoA reductase, SMT2, or14alpha demethylase) in a tobacco and at least one PAH upon pyrolysis ofsaid tobacco.

Some of these nucleic acid embodiments comprise, consist, or consistessentially of fragments of the QPTase, PMTase, and A622 genes that werefound to inhibit gene expression unexpectedly well in the RNAiconstructs described herein, producing reduced alkaloid tobacco (below7,000 ppm, 1,000 ppm, or 500 ppm). Some of these nucleic acids concernfragments of genes involved in sterol biosynthesis (e.g., squalenesynthase, HMG-CoA reductase, SMT2, or 14alpha demethylase) and thesefragments are particularly useful for inhibiting production of sterolsin tobacco and PAHs when said tobacco undergoes pyrolysis.

Still more of the nucleic acid embodiments concern several phytoenedesaturase (PDS) mutants (e.g., PDSM-1, PDSM-2, and PDSM-3, SEQ. ID.NOs.: 10, 11, or 12) that were developed to confer resistance tonorflurazone, which allows both tissue-culture selection of cellstransformed with the construct, as well as, field-based selection,wherein weeds and tobacco, which do not contain an herbicide resistancegene, are removed from the field or crop by spraying the herbicidenorflurazone or an herbicide of the same class or activity (e.g.,herbicides that contain C₁₂H₉ClF₃N₃O (see U.S. Pat. No. 3,644,355,herein expressly incorporated by reference in its entirety), but plantsexpressing PDSM-1, PDSM-2, or PDSM-3 survive the herbicide contact).That is, some embodiments include isolated nucleic acids that comprise,consist, or consist essentially of the PDS mutant sequences provided bySEQ. ID. NOs.:10, 11, or 12 and fragments thereof at least 30nucleotides in length (e.g., less than, greater than or equal to 30, 35,40, 45, 50, 60, 75, 100, 150, 250, 500, 750, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, or 1729 consecutive nucleotides) that include amutation (e.g., T1478G, which encodes Val493Gly; G863C, which encodesArg288Pro; and T1226C, which encodes Leu409Pro) that confers resistanceto norflurazone). Preferably, the fragments of the PDS mutants describedherein confer resistance to norflurazone, although fragments that do notconfer resistance to the herbicide are also useful in the field inassays designed to follow the retention of constructs described hereinin successive generations of transgenic plants. Approaches to developmore norflurazone-resistance genes are also provided herein.

Additional embodiments include isolated nucleic acids that comprise,consist, or consist essentially of root-specific promoters, constitutivepromoters, and developmentally regulated promoters, which can be usedinterchangeably with the nucleic acid sequences described herein. Someembodiments, for example, include a root-specific promoter such as theRD2 promotor (SEQ. ID NO. 37 or SEQ. ID NO. 50), truncated RD2 promoter(SEQ. ID NO. 13) or the Putrescene methyl transferase promoter (PMT-1)(SEQ. ID NO. 14). Constitutive promoters that can be used withembodiments described herein include the GapC promoter (SEQ. ID. NO.:15), Actin 2 promoter (Act2P) (SEQ. ID NO. 16), the tobacco alcoholdehydrogenase promoter (ADP) (SEQ. ID NO. 17), the Arabidopsis ribosomalprotein L2 promoter (RPL2P) (SEQ. ID NO. 18), and the nopaline synthasepromoter (NOS P) (SEQ ID NO. 46). Developmentally regulated promotersthat can be used with the nucleic acid sequences described hereininclude the cinnamyl alcohol dehydrogenase promoter (SEQ. ID NO. 19) andthe metallothionein I promoter (SEQ. ID NO. 20). Additional embodimentsalso include isolated nucleic acids that comprise, consist, or consistessentially of the GAD2 terminator (SEQ. ID NO. 21), nopaline synthaseterminator (NOS T) (SEQ ID NO 38), a FAD2 intron (provided by (SEQ. IDNO. 22), ACT11 intron 3 (SEQ ID NO 41), which was used as a spacer inseveral of the RNAi constructs, and the PAP1 intron (provided bynucleotides 6446-7625 of (SEQ. ID NO. 33). Because of the uniqueproperties of the FAD2 intron, in particular the hair-pin secondarystructure afforded by the interaction of splice sites in the sequence,it was found, unexpectedly, that transgenic tobacco could be made withvarious inhibitory sequences with nearly equivalent success (e.g.,approximately 50% of the reduced nicotine lines created by multipleconstructs were found to have less than 1,000 ppm total alkaloid).Accordingly, significantly improved RNAi constructs were generated usingthis spacer. That is, embodiments provided herein concern the use of anintronic sequence comprising splicing recognition sequences (preferablyFAD2 or PAP1 intron) to link or join a first RNA sequence to a secondRNA sequence that is complementary to said first RNA sequence, whereinsaid first or second RNA sequence is complementary to a target RNA,which, preferably, regulates the production of a harmful compound intobacco (e.g., nicotine, nornicotine, or a sterol).

Embodiments provided herein also concern isolated nucleic acids thatcomprise, consist, or consist essentially of the inhibition andselection cassettes identified as SEQ. ID. Nos. 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 and fragments thereof (e.g., a fragment that is atleast, less than or equal to or greater than 30, 40, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640,660, 680, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100,4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300,5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500,6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700,7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900,or 9000 consecutive nucleotides) of SEQ. ID. Nos. 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49 or 50).

Embodiments provided herein also concern isolated nucleic acids thatcomprise, consist, or consist essentially of a plurality of the nucleicacid sequences described herein. For example, a double knock-outconstruct comprising a portion of the A622 gene and a portion of theQPTase gene has been made and it is expected that this construct willefficiently reduce expression of at least two genes involved in thesynthesis or regulation of the production of nicotine (SEQ. ID. No. 27).Another double knock-out construct comprises, consists of or consistsessentially of a first isolated nucleic acid that inhibits nicotinebiosynthesis (e.g., A622) and a second isolated nucleic acid thatinhibits synthesis of at least one sterol (e.g., SMT2). (See (SEQ. ID.No. 33)). Accordingly, embodiments provided herein concern an isolatednucleic acid construct that inhibits the expression of a plurality ofgenes that regulate the production of more than one harmful compound intobacco. In some aspects of these embodiments, said isolated nucleicacid construct inhibits the expression of at least two nicotinebiosynthesis genes, a nicotine biosynthesis gene and a sterolbiosynthesis gene, or two sterol biosynthesis genes. It should also beunderstood that embodiments provided herein concern tobacco generated bycrossing the transgenic tobaccos described herein. For example, someembodiments concern progeny of a cross between a transgenic tobaccohaving a reduced amount of nicotine and a transgenic tobacco having areduced amount of a sterol. Crossings of the transgenic tobaccodescribed herein and wild-type tobacco are also embodiments providedherein.

The interfering RNAs used with the embodied nucleic acids can beexpressed from nucleic acid construct that encodes one or more strandsof the RNA duplex of the interfering RNA. In some embodiments, thenucleic acid construct is present on a vector. The vectors may be viralvectors, plasmids, or any other vehicles for nucleic acid delivery. Inother embodiments, the interfering RNAs described herein can begenerated synthetically by methods, such as direct synthesis or in vitrotranscription. In some embodiments, synthetic interfering nucleic acidscomprising modified nucleic acids are contemplated. Other embodimentsprovided herein include multiple vector systems for producing aninterfering RNA wherein a first vector encodes the first strand of theinterfering RNA and a second vector encodes the second strand of theinterfering RNA.

Still other embodiments provided herein relate to tobacco cellscomprising one or more of the nucleic acid constructs described herein,which encode an interfering RNA that is specific for a gene productinvolved in nicotine or sterol biosynthesis. In such embodiments, theinterfering RNA reduces or eliminates the expression of such geneproduct. Additional embodiments relate to tobacco cells comprising oneor more interfering RNAs that are specific for a gene product involvedin nicotine biosynthesis. In certain embodiments, the interfering RNAsare synthetic interfering RNAs.

Certain embodiments provided herein relate to tobacco plants and curedtobacco products having a reduced amount or nicotine, nornicotine,TSNAs, and/or sterols. In such embodiments, reduction in nicotine,nornicotine, TSNAs, and/or sterol amounts in the tobacco plants andcured tobacco products is mediated by an interfering RNA comprising anRNA duplex wherein at least 30 consecutive nucleotides (e.g., at leastor equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480,500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000consecutive nucleotides) of the RNA duplex are complementary orsubstantially complementary to a target mRNA that encodes a gene productinvolved in nicotine biosynthesis. Further aspects relate to a field orcrop of tobacco plants comprising one or more of the constructsdescribed herein. Still other aspects relate to a tobacco seed producedfrom one or more of the tobacco plants provided herein.

Transgenic tobacco plants produced by the methods described herein canbe cured by any of the tobacco curing techniques that are known in theart. As such, some embodiments provided herein relate to cured tobaccoand cure tobacco products made from the transgenic plants describedherein. In some embodiments, the cured tobacco product is a blendedtobacco product. In some embodiments, the cured tobacco product isprocessed in a microbe-free environment. In other embodiments, the curedtobacco is contacted with sterilizing vapor, heat, or radiation so as toprevent the conversion of alkaloid to TSNAs.

Some embodiments provided herein relate to methods of preparing atobacco cell having a reduced nicotine and/or sterol content, whereinthe method comprises providing a tobacco cell with one or moreinterfering RNAs or one or more nucleic acid constructs encoding aninterfering RNA comprising an RNA duplex, which comprises a first strandhaving a sequence substantially similar or identical to at least aportion of the coding sequence of a target gene and/or target geneproduct involved in nicotine and/or sterol biosynthesis, and a secondstrand that is complementary or substantially complementary to the firststrand. In a preferred embodiment, the target gene product involved innicotine biosynthesis is QTPase, PMTase, or A622 and the target geneproduct involved in sterol biosynthesis is squalene synthase, HMG-CoAreductase, SMT2, or 14alpha demethylase.

Other embodiments provided herein relate to methods of preparing atobacco plant having a reduced nicotine and/or sterol content comprisingobtaining a tobacco cell in culture; providing to the tobacco cell oneor more interfering RNAs or one or more nucleic acid constructs encodingan interfering RNA comprising an RNA duplex, which comprises a firststrand having a sequence substantially similar or identical to at leasta portion of the coding sequence of a target gene and/or target geneproduct involved in nicotine and/or sterol biosynthesis, and a secondstrand that is complementary or substantially complementary to the firststrand; allowing expression of the interfering RNA, thereby reducingcellular nicotine and/or sterol content; and regenerating a tobaccoplant from the tobacco cell. In some embodiments, the tobacco plantsprepared by such method also have a reduced TSNA content and/or producea reduced amount of PAHs upon pyrolysis, as compared to a conventionaltobacco product of the same class, a reference tobacco product (e.g.,IM16), or the same strain of tobacco prior to genetic modification.

As mentioned above, additional embodiments include tobacco products thathave been carefully blended so that desired levels of nicotine, TSNAs,and/or sterols are obtained. For example, tobacco having a reduced levelof nicotine and/or TSNAs, prepared as described above, can be blendedwith conventional tobacco so as to obtain virtually any amount ofnicotine and/or sterols. Additionally, as mentioned above, exogenousnicotine can be added to the tobacco or tobacco product. Further, two ormore varieties of tobacco (e.g., transgenic reduced alkaloid Burley,transgenic reduced alkaloid Flue-cured, and/or transgenic reducedalkaloid Oriental) can be blended so as to achieve a desired taste whilemaintaining nicotine levels in or delivered by the product (e.g., asmeasured by FTC methodology) at less than 7,000 ppm, 5,000 ppm, 3000ppm, 2000 ppm, 1000 ppm, or 500 ppm and TSNA levels at 0.5 μg/g or less.Similarly, two or more varieties of transgenic tobacco having a reducedamount of sterols can be blended, as above, or varieties ofsterol-reduced transgenic tobacco can be blended with varieties ofnicotine reduced transgenic tobacco. In this manner, differences invariety, flavor, as well as amounts of nicotine and/or sterols can beincrementally adjusted. These blended tobaccos can be processed intotobacco products, which can be incorporated into tobacco use cessationkits (e.g., a multiple step nicotine reduction program, whereby aconsumer's exposure to nicotine, TSNA, or PAH is gradually reduced overtime by consumption of tobacco products that have increasingly smallerquantities of these compounds). Such kits and programs, are designed toreduce or eliminate nicotine dependence and reduce the potential tocontribute to a tobacco related disease.

More embodiments concern methods to reduce the carcinogenic potential oftobacco products, including cigarettes, cigars, chewing tobacco, snuffand tobacco-containing gum and lozenges. Some methods, for exampleinvolve the use of the constructs described herein to obtain transgenictobacco that comprises a reduced amount of nicotine, TSNAs, and/orsterols and the manufacture of tobacco products containing said tobacco.Accordingly, the transgenic tobacco plants, described above, areharvested, cured, and processed into tobacco products. These tobaccoproducts have a reduced carcinogenic potential because they are preparedfrom tobacco that has a reduced amount of nicotine, TSNAs, and sterols.Smoke or smoke condensate generated from these tobaccos and tobaccoproducts can also be evaluated using the assays provided herein so as toconfirm that said tobaccos and tobacco products have a reduced potentialto contribute to a tobacco-related disease and that said tobaccos andtobacco products are reduced risk compositions.

Yet another aspect provided herein concerns the reduction of the amountof TSNAs, preferably NNN and NNK, and polyaromatic hydrocarbons (PAHs),preferably, benz[a]pyrene and metabolites thereof in humans who smoke,consume or otherwise ingest tobacco. This method is practiced byproviding a tobacco product comprising a transgenic tobacco thatcomprises a reduced amount of nicotine and/or a sterol to said humans,thereby lowering the amount of TSNAs and/or PAHs in said humans exposedto said tobacco product. By one approach, for example, the carcinogenicpotential of side stream or main stream tobacco smoke in a human exposedto said side stream or main stream tobacco smoke is reduced by providingthe cured tobacco as described above in a product that undergoespyrolysis, wherein pyrolysis of said product results in side stream ormain stream smoke comprising a reduced amount of TSNAs and/or PAHs. Thesection below describes several preferred approaches to developgenetically modified tobaccos and tobacco products containinggenetically modified tobacco that have a reduced amount of a compoundthat contributes to a tobacco related disease.

Preparation of Preferred Transgenic Tobaccos

A first generation of transgenic Burley tobacco was created using afull-length antisense QPTase construct. Tobacco of the variety Burley 21LA was transformed with the binary Agrobacterium vector pYTY32 toproduce a low nicotine tobacco variety, Vector 21-41. The binary vectorpYTY32 carried the 2.0 kb NtQPT1 root-cortex-specific promoter drivingantisense expression of the NtQPT1 cDNA (SEQ. ID. NO. 2) and thenopaline synthase (nos) 3′ termination sequences from Agrobacteriumtumefaciens T-DNA. The selectable marker for this construct was neomycinphosphotransferase (nptII) from E. coli Tn5 which confers resistance tokanamycin, and the expression nptII was directed by the nos promoterfrom Agrobacterium tumefaciens T-DNA. Transformed cells, tissues, andseedlings were selected by their ability to grow on Murashige-Skoog (MS)medium containing 300 μg/ml kanamycin. Burley 21 LA is a variety ofBurley 21 with substantially reduced levels of nicotine as compared withBurley 21 (i.e., Burley 21 LA has 8% the nicotine levels of Burley 21,see Legg et al., Can J Genet Cytol, 13:287-91 (1971); Legg et al., JHered, 60:213-17 (1969)).

One-hundred independent pYTY32 transformants of Burley 21 LA (To) wereallowed to self. Progeny of the selfed plants (T₁) were germinated onmedium containing kanamycin and the segregation of kanamycin resistancescored. T₁ progeny segregating 3:1 resulted from transformation at asingle locus and were subjected to further analysis.

Nicotine levels of T₁ progeny segregating 3:1 were measuredqualitatively using a micro-assay technique. Approximately ˜200 mg freshtobacco leaves were collected and ground in 1 ml extraction solution(Extraction solution: 1 ml Acetic acid in 100 ml H₂O). Homogenate wascentrifuged for 5 min at 14,000×g and supernatant removed to a cleantube, to which the following reagents were added: 100 μL NH₄OAC (5 g/100ml H₂O+50 μL Brij 35); 500 μL Cyanogen Bromide (Sigma C-6388, 0.5 g/100ml H₂O+50 μL Brij 35); 400 μL Aniline (0.3 ml buffered Aniline in 100 mlNH₄OAC+50 μL Brij 35). A nicotine standard stock solution of 10 mg/ml inextraction solution was prepared and diluted to create a standard seriesfor calibration. Absorbance at 460 nm was read and nicotine content oftest samples were determined using the standard calibration curve.

T₁ progeny that had less than 10% of the nicotine levels of the Burley21 LA parent were allowed to self to produce T₂ progeny. Homozygous T₂progeny were identified by germinating seeds on medium containingkanamycin and selecting clones in which 100% of the progeny wereresistant to kanamycin (i.e., segregated 4:0; heterozygous progeny wouldsegregate 3:1). Nicotine levels in homozygous and heterozygous T₂progeny were qualitatively determined using the micro-assay and againshowed levels less than 10% of the Burley 21 LA parent. Leaf samples ofhomozygous T₂ progeny were sent to the Southern Research and TestingLaboratory in Wilson, N.C. for quantitative analysis of nicotine levelsusing Gas Chromatography/Flame Ionization Detection (GC/FID). HomozygousT₂ progeny of transformant #41 gave the lowest nicotine levels (˜70ppm), and this transformant was designated as “Vector 21-41.”

Vector 21-41 plants were allowed to self-cross, producing T₃ progeny. T₃progeny were grown and nicotine levels assayed qualitatively andquantitatively. T₃ progeny were allowed to self-cross, producing T₄progeny. Samples of the bulked seeds of the T₄ progeny were grown andnicotine levels tested.

In general, Vector 21-41 is similar to Burley 21 LA in all assessedcharacteristics, with the exception of alkaloid content and totalreducing sugars (e.g., nicotine and nor-nicotine). Vector 21-41 may bedistinguished from the parent Burley 21 LA by its substantially reducedcontent of nicotine, nor-nicotine and total alkaloids. As shown below,total alkaloid concentrations in Vector 21-41 are significantly reducedto approximately relative to the levels in the parent Burley 21 LA, andnicotine and nor-nicotine concentrations show dramatic reductions inVector 21-41 as compared with Burley 21 LA. Vector 21-41 also hassignificantly higher levels of reducing sugars as compared with Burley21 LA.

Field trials of Vector 21-41 T₄ progeny were performed at the CentralCrops Research Station (Clayton, N.C.) and compared to the Burley 21 LAparent. The design was three treatments (Vector 21-41, a Burley 21 LAtransformed line carrying only the NtQPT1 promoter [Promoter-Control],and untransformed Burley 21 LA [Wild-type]), 15 replicates, 10 plantsper replicate. The following agronomic traits were measured andcompared: days from transplant to flowering; height at flowering; leafnumber at flowering; yield; percent nicotine; percent nor-nicotine;percent total nitrogen; and percent reducing sugars.

Vector 21-41 was also grown on approximately 5000 acres by greater than600 farmers in five states (Pennsylvania, Mississippi, Louisiana, Iowa,and Illinois). The US Department of Agriculture, Agriculture MarketingService (USDA-AMS) quantified nicotine levels (expressed as percentnicotine per dry weight) using the FTC method of 2,701 samples takenfrom these farms. Nicotine levels ranged from 0.01% to 0.57%. Theaverage percent nicotine level for all these samples was 0.09%, with themedian of 0.07%. Burley tobacco cultivars typically have nicotine levelsbetween 2% and 4% dry weight (Tso, T. C., 1972, Physiology andBiochemistry of Tobacco Plants. Dowden, Hutchinson, and Ross, Inc.Stroudsbury).

A transgenic Flue-cured tobacco with a reduced amount of nicotine andTSNAs was created using an RNAi approach. FIG. 1 illustrates an RNAiconstruct that was used to create a reduced nicotine tobacco, whereinthe root-specific promoter RD2 (Bp 1-2010) was used to drive expressionof an RNAi cassette comprising an antisense full-length QPTase cDNA (Bp2011-3409) linked to a 382 bp fragment of the cucumber aquaporin gene(Bp 3410-3792), which is linked to a sense full-length QPTase cDNA (Bp3793-5191) and the GapC terminator (Bp5192-5688) (see SEQ. ID. No. 23).This first RNAi construct also comprises a GUS-selection cassettecomprising the GapC promoter (Bp 1-1291), which drives expression of theGUS gene (Bp 1292-3103), linked to the GapC terminator (Bp 3104-3600)(see SEQ. ID. No. 34). This first RNAi construct was ligated into abinary vector, pBin19 which was then introduced into Agrobacteriumtumefaciens. Leaf disks from Flue-cured variety K326 were thentransformed with Agrobacterium that contained the RNAi constructcomprising the RNAi cassette and the GUS selection cassette. GUS-basedselection was then employed to select positively transformed plantlets(buds), which were then regenerated to plants. Leaf samples were thenharvested and the alkaloid content was then determined. The alkaloidcontent of samples obtained from some of the transgenic lines createdwith this first RNAi construct was 6000 ppm. Since the total alkaloidcontent in tobacco is about 90% nicotine, it is understood by thoseskilled in the art that the transgenic Flue-cured tobacco created usingthe construct shown in FIG. 1 has significantly reduced levels ofnicotine and TSNA, as compared to a conventional tobacco, a referencetobacco, or the parental strain of tobacco prior to geneticmodification. Accordingly, tobacco products (e.g., cigarettes), tobacco,tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured orOriental comprising this RNAi construct are embodiments provided herein.

FIG. 2 shows another RNAi construct that was used to generate severallines of reduced nicotine and TSNA tobacco. This RNAi construct has aQTPase inhibition cassette (SEQ. ID. No. 24) and a norflurazoneselection cassette (SEQ. ID. No. 35). Starting from the right border(RB), the QPTase inhibition cassette comprises an RD2 promoter (Bp1-2010) operably linked to an antisense fragment (360 bp) (Bp 2011-2370)of the QTPase gene, joined to a FAD2 intron (Bp 2371-3501), which isjoined to a sense fragment of the QTPase gene (360 bp) (Bp 3502-3861),which is joined to the GAD2 terminator (Bp 3862-4134). The selectioncassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to amutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to theGapC terminator (Bp 2891-3387) at the left border (LB). Accordingly,tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobaccocells, tobacco seeds, in Burley, Flue-cured or Oriental comprising thisRNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 2using Agrobacterium-mediated transformation and 1,140 independent lineswere selected, regenerated, and transplanted in the greenhouse. Of the1, 140 independent lines, 1,097 plants were harvested and tested foralkaloid content. A total of 608 lines were identified as having lessthan 1,000 ppm total alkaloid and 139 lines were identified as havingless than 500 ppm total alkaloid. Accordingly, the transgenic Flue-curedtobacco created using the construct shown in FIG. 2 has significantlyreduced levels of nicotine and TSNA, as compared to a conventionaltobacco, a reference tobacco, or the parental strain of tobacco prior togenetic modification.

Burley tobacco was also transformed with the construct shown in FIG. 2using Agrobacterium-mediated transformation and 385 independent lineswere selected, regenerated, and transplanted in the greenhouse. Of the385 independent lines, 350 lines of plants were harvested and tested foralkaloid content. A total of 142 lines were identified as having lessthan 1,000 ppm total alkaloid and 10 lines were identified as havingless than 500 ppm total alkaloid. Accordingly, it is understood by thoseskilled in the art that the transgenic Burley tobacco created using theconstruct shown in FIG. 2 also has significantly reduced levels ofnicotine and TSNA, as compared to a conventional tobacco, a referencetobacco, or the parental strain of tobacco prior to geneticmodification.

Oriental tobacco will be transformed with the construct shown in FIG. 2using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Oriental tobacco that will be created using the constructshown in FIG. 2 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

FIG. 3 illustrates another RNAi construct that can be used to create areduced nicotine and TSNA transgenic tobacco. This RNAi construct has aPMTase inhibition cassette (SEQ. ID. No. 25) and a norflurazoneselection cassette (SEQ. ID. No. 35). Starting from the right border(RB), the PMTase inhibition cassette comprises an RD2 promoter (Bp1-2010) operably linked to an antisense nucleic acid (241 bp) (Bp2011-2251) of a PMTase gene, joined to a FAD2 intron (Bp 2252-3382),which is joined to a sense nucleic acid of the PMTase gene (241 bp) (Bp3383-3623), which is joined to the GAD2 terminator (Bp 3624-3896). Theselection cassette comprises the Actin 2 promoter (Bp 1-1161) operablylinked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890)joined to the GapC terminator (Bp 2891-3387) at the left border (LB).Accordingly, tobacco products (e.g., cigarettes), tobacco, tobaccoplants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Orientalcomprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the construct shown in FIG.3 using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Flue-cured tobacco that will be created using theconstruct shown in FIG. 3 will have significantly reduced levels ofnicotine and TSNA, as compared to a conventional tobacco, a referencetobacco, or the parental strain of tobacco prior to geneticmodification.

Burley tobacco will be transformed with the construct shown in FIG. 3using Agrobacterium-mediated, Transbacter-mediated (see e.g.,Broothaerts et al., Nature 433:629 (2005), herein expressly incorporatedby reference in its entirety) or biolistic transformation andindependent lines will be selected, regenerated, and transplanted in thegreenhouse. Most of the independent lines grown in the greenhouse willbe harvested and tested for alkaloid content. It is expected thatapproximately 50% of the lines tested will have less than 1,000 ppmtotal alkaloid and approximately 10% of the lines tested will have lessthan 500 ppm total alkaloid. Accordingly, it is expected that thetransgenic Burley tobacco that will be created using the construct shownin FIG. 3 will have significantly reduced levels of nicotine and TSNA,as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown inFIG. 3 using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Oriental tobacco that will be created using the constructshown in FIG. 3 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

FIG. 4 illustrates another RNAi construct that was used to create areduced nicotine and TSNA transgenic tobacco. This RNAi construct has aA622 inhibition cassette (SEQ. ID. No. 26) and a norflurazone selectioncassette (SEQ. ID. No. 35). Starting from the right border (RB), theA622 inhibition cassette comprises an RD2 promoter (Bp 1-2010) operablylinked to an antisense nucleic acid (628 bp) (Bp 2011-2638) of the A622gene, joined to a FAD2 intron (Bp 2639-3769), which is joined to a sensenucleic acid of the A622 gene (628 bp) (Bp 3770-4397), which is joinedto the GAD2 terminator (Bp 4398-4670). The selection cassette comprisesthe Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoenedesaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp2891-3387) at the left border (LB). Accordingly, tobacco products (e.g.,cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, inBurley, Flue-cured or Oriental comprising this RNAi construct areembodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 4using Agrobacterium-mediated transformation and 270 independent lineswere selected, regenerated, and transplanted in the greenhouse. Of the270 independent lines, 259 plants were harvested and tested for alkaloidcontent. A total of 131 lines were identified as having less than 1,000ppm total alkaloid and 45 lines were identified as having less than 500ppm total alkaloid. Accordingly, it is understood by those skilled inthe art that the transgenic Flue-cured tobacco created using theconstruct shown in FIG. 4 also has significantly reduced levels ofnicotine and TSNA, as compared to a conventional tobacco, a referencetobacco, or the parental strain of tobacco prior to geneticmodification.

Several lines that were transformed with this construct wereunexpectedly found to have conventional levels of nicotine but asignificantly reduced amount of nornicotine. That is, 9 lines were foundto have nicotine levels ranging from 2.17 mg/g to 3.99 mg/g andnornicotine levels less than or equal to 0.00 to 0.06 mg/g (see Table2).

TABLE 2 Transgenic tobacco having reduced nornicotine and conventionalamounts of nicotine Alkaloid Nornicotine Nicotine new I.D (ppm) (mg/g)(mg/g) VDG 020 2486.53 0.00 2.30 VDG 032 4683.01 0.00 3.48 VDG 0454490.79 0.00 3.94 VDG 052 2855.58 0.00 2.61 VDG 054 2291.89 0.00 2.17VDG 077 4857.86 0.06 3.99 VDG 097 3072.40 0.00 2.58 VDG 107 4921.31 0.033.59 VDG 116 4960.64 0.00 3.56 Control-8 5005.22 0.28 4.02 Control-205711.97 0.34 5.35 Control-28 5196.25 0.24 4.52 *Highlighted entries showtransgenic tobacco lines having a reduced amount of nornicotine andconventional amounts of nicotine.

Tobacco products containing the selectively reduced nornicotinetransgenic tobacco described above are also embodiments provided herein.That is, tobacco products comprising a transgenic tobacco that comprisesa conventional amount of nicotine (e.g., comprise or delivers accordingto FTC methodology at least, less than, greater than, or equal to 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/gnicotine) and a reduced amount of nornicotine (e.g., 0.00, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14,0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg/g), as compared to aconventional tobacco, a reference tobacco, or the parental strain oftobacco prior to genetic modification, are embodiments provided herein.Particularly preferred are transgenic tobacco and tobacco products madetherefrom, which comprise a conventional amount of nicotine (e.g.,comprises or delivers by FTC methodology at least, less than, greaterthan, or equal to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,4.9, or 5.0 mg/g nicotine) and a reduced amount of nornicotine (e.g.,0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11,0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg/g), ascompared to a conventional tobacco, a reference tobacco, or the parentalstrain of tobacco prior to genetic modification, and an isolatedfragment of the A622 gene, in particular, comprising, consisting of, orconsisting essentially of an isolated nucleic acid of SEQ. ID. No. 5, orthe cassette of SEQ. ID. No. 26.

Burley tobacco will be transformed with the construct shown in FIG. 4using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Burley tobacco that will be created using the constructshown in FIG. 4 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification. It is alsoexpected that some lines of tobacco created with the afore-mentionednucleic acid construct will retain conventional amounts of nicotine butwill comprise a reduced amount of nornicotine, as compared to aconventional tobacco, a reference tobacco, or the parental strain oftobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown inFIG. 4 using Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Oriental tobacco that will be created using the constructshown in FIG. 4 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification. It is alsoexpected that some lines of tobacco created with the afore-mentionednucleic acid construct will retain conventional amounts of nicotine butwill comprise a reduced amount of nornicotine, as compared to aconventional tobacco, a reference tobacco, or the parental strain oftobacco prior to genetic modification.

FIG. 5 illustrates a double-knock-out RNAi construct, which has beencreated to develop a reduced nicotine and TSNA transgenic tobacco. Thisdouble-knock-out RNAi construct has a QPTase/A622 inhibition cassette(SEQ. ID. No. 27) and a norflurazone selection cassette (SEQ. ID. No.35). Starting from the right border (RB), the QPTase/A622 inhibitioncassette comprises an RD2 promoter (Bp 1-2010) operably linked to aQPTase antisense nucleic acid (360 bp) (Bp 2011-2370) of a QPTase gene,which is joined to a A622 antisense nucleic acid (628 bp) (Bp 2371-2998)of a A622 gene, which is joined to a FAD2 intron (Bp 2999-4129), whichis joined to a sense nucleic acid of the A622 gene (628 bp) (Bp4130-4757), which is joined to a sense nucleic acid of the QPTase gene(360 bp) (Bp 4758-5117), which is joined to the GAD2 terminator (Bp5118-5390). The selection cassette comprises the Actin 2 promoter (Bp1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp1162-2890) joined to the GapC terminator (Bp 2891-3387) at the leftborder (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco,tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured orOriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the construct shown in FIG.5 using Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Flue-cured tobacco that will be created using theconstruct shown in FIG. 5 will have significantly reduced levels ofnicotine and TSNA, as compared to a conventional tobacco, a referencetobacco, or the parental strain of tobacco prior to geneticmodification.

Burley tobacco will be transformed with the construct shown in FIG. 5using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Burley tobacco that will be created using the constructshown in FIG. 5 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown inFIG. 5 using Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Oriental tobacco that will be created using the constructshown in FIG. 5 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

More embodiments concern an RNAi construct designed to reduce the amountof sterols in tobacco and thereby reduce production of a PAH uponpyrolysis of said transgenic tobacco. A first sterol-reducing RNAiconstruct has a 14alpha demethylase inhibition cassette (SEQ. ID. No.28). The 14alpha demethylase inhibition cassette comprises a double (twopromoters in tandem) 35S promoter (Bp 1-618) operably linked to anantisense 14alpha demethylase nucleic acid (Bp 619-1503), which isjoined to a FAD2 intron (Bp 1504-2634), which is joined to a sensenucleic acid of the 14alpha demethylase gene (Bp 2635-3519), which isjoined to the Nos terminator (Bp 3520-3773). Accordingly, tobaccoproducts (e.g., cigarettes), tobacco, tobacco plants, tobacco cells,tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAiconstruct are embodiments provided herein.

Flue-cured tobacco will be transformed with the 14alpha demethylaseinhibition cassette using Agrobacterium-mediated, Transbacter-mediated,or biolistic transformation and independent lines will be selected,regenerated, and transplanted in the greenhouse. Most of the independentlines grown in the greenhouse will be harvested and tested for sterolcontent. It is expected that approximately 50% of the lines tested willhave significantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Flue-cured tobacco thatwill be created using the construct above will have significantlyreduced levels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the 14alpha demethylaseinhibition cassette using Agrobacterium-mediated, Transbacter-mediated,or biolistic transformation and independent lines will be selected,regenerated, and transplanted in the greenhouse. Most of the independentlines grown in the greenhouse will be harvested and tested for sterolcontent. It is expected that approximately 50% of the lines tested willhave significantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Burley tobacco that willbe created using the construct above will have significantly reducedlevels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the 14alpha demethylaseinhibition cassette using Agrobacterium-mediated, Transbacter-mediated,or biolistic transformation and independent lines will be selected,regenerated, and transplanted in the greenhouse. Most of the independentlines grown in the greenhouse will be harvested and tested for sterolcontent. It is expected that approximately 50% of the lines tested willhave significantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Oriental tobacco thatwill be created using the construct above will have significantlyreduced levels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

More embodiments concern another RNAi construct designed to reduce theamount of a sterol in tobacco and thereby reduce production of a PAHupon pyrolysis of said transgenic tobacco. A second sterol-reducing RNAiconstruct has a SMT2 inhibition cassette (SEQ. ID. No. 29). The SMT2inhibition cassette comprises a double (two promoters in tandem) 35Spromoter (Bp 1-618) operably linked to an antisense SMT2 nucleic acid(Bp 619-1398), which is joined to a FAD2 intron (Bp 1399-2529), which isjoined to a sense nucleic acid of the SMT2 gene (Bp 2530-3309), which isjoined to the Nos terminator (Bp 3310-3563). Accordingly, tobaccoproducts (e.g., cigarettes), tobacco, tobacco plants, tobacco cells,tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAiconstruct are embodiments provided herein.

Flue-cured tobacco will be transformed with the SMT2 inhibition cassetteusing Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for sterol content. It isexpected that approximately 50% of the lines tested will havesignificantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Flue-cured tobacco thatwill be created using the construct above will have significantlyreduced levels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the SMT2 inhibition cassetteusing Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for sterol content. It isexpected that approximately 50% of the lines tested will havesignificantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Burley tobacco that willbe created using the construct above will have significantly reducedlevels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the SMT2 inhibition cassetteusing Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for sterol content. It isexpected that approximately 50% of the lines tested will havesignificantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Oriental tobacco thatwill be created using the construct above will have significantlyreduced levels of sterols and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

More embodiments concern another RNAi construct designed to reduce theamount of a sterol in tobacco and thereby reduce production of a PAHupon pyrolysis of said transgenic tobacco. A third sterol-reducing RNAiconstruct has a squalene synthase inhibition cassette (SEQ. ID. No. 30).The squalene synthase inhibition cassette comprises a double (twopromoters in tandem) 35S promoter (Bp 1-618) operably linked to anantisense squalene synthase nucleic acid (Bp 619-1057), which is joinedto a FAD2 intron (Bp 1058-2188), which is joined to a sense nucleic acidof the squalene synthase gene (Bp 2189-2627), which is joined to the Nosterminator (Bp 2628-2881). Accordingly, tobacco products (e.g.,cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, inBurley, Flue-cured or Oriental comprising this RNAi construct areembodiments provided herein.

Flue-cured tobacco will be transformed with the squalene synthaseinhibition cassette using Agrobacterium-mediated, Transbacter-mediated,or biolistic transformation and independent lines will be selected,regenerated, and transplanted in the greenhouse. Most of the independentlines grown in the greenhouse will be harvested and tested for sterolcontent. It is expected that approximately 50% of the lines tested willhave significantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Flue-cured tobacco thatwill be created using the construct above will have significantlyreduced levels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the squalene synthase inhibitioncassette using Agrobacterium-mediated, Transbacter-mediated, orbiolistic transformation and independent lines will be selected,regenerated, and transplanted in the greenhouse. Most of the independentlines grown in the greenhouse will be harvested and tested for sterolcontent. It is expected that approximately 50% of the lines tested willhave significantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Burley tobacco that willbe created using the construct above will have significantly reducedlevels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the squalene synthaseinhibition cassette using Agrobacterium-mediated, Transbacter-mediated,or biolistic transformation and independent lines will be selected,regenerated, and transplanted in the greenhouse. Most of the independentlines grown in the greenhouse will be harvested and tested for sterolcontent. It is expected that approximately 50% of the lines tested willhave significantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Oriental tobacco thatwill be created using the construct above will have significantlyreduced levels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

More embodiments concern yet another RNAi construct designed to reducethe amount of a sterol in tobacco and thereby reduce production of a PAHupon pyrolysis of said transgenic tobacco. A fourth sterol-reducing RNAiconstruct has a HMG-CoA reductase inhibition cassette (SEQ. ID. No. 31).The HMG-CoA reductase inhibition cassette comprises a double (twopromoters in tandem) 35S promoter (Bp 1-618) operably linked to anantisense HMG-CoA reductase nucleic acid (Bp 619-1468), which is joinedto a FAD2 intron (Bp 1469-2599), which is joined to a sense nucleic acidof the HMG-CoA reductase gene (Bp 2600-3449), which is joined to the Nosterminator (Bp 3450-3703). Accordingly, tobacco products (e.g.,cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, inBurley, Flue-cured or Oriental comprising this RNAi construct areembodiments provided herein.

Flue-cured tobacco (K326) was transformed with the HMG-CoA reductaseinhibition cassette using Agrobacterium-mediated transformation andindependent lines were selected, regenerated, and transplanted in thegreenhouse. Several independent lines grown in the greenhouse wereharvested and tested for the presence of various sterols (see Table 3).As shown in the table, several lines (e.g., HMGIR 1, HMGIR 2, HMGIR 3-2,HMGIR 4, HMGIR 7, HMGIR 11, HMGIR 13, HMGIR 16, HMGIR 18, HMGIR 19) werefound to have significantly reduced levels of sterols, as compared tothe parental strain of tobacco (i.e., tobacco of the same variety priorto genetic modification). Accordingly, embodiments include transgenictobacco and tobacco products made therefrom comprising a reduced amountof sterols, as compared to a tobacco of the same variety, parentalstrain or a tobacco that has not been genetically modified. It isexpected that the transgenic Flue-cured tobacco that was created usingthe construct above will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

TABLE 3 HmgCoa Reductase inhibition K 326 HMGIR HMGIR HMGIR HMGIR HMGIRHMGIR HMGIR HMGIR HMGIR HMGIR cont 1 2 3-2 4 7 11 13 16 18 19 Squalene 11.47 0.90 1.96 2.64 1.00 1.25 1.21 0.72 0.90 0.75 Squalene 1 1.48 0.882.13 2.78 0.94 1.14 1.12 0.97 0.73 0.96 Tocopherol 1 1.67 2.02 1.15 1.401.13 1.69 1.15 1.36 1.48 1.13 Tocopherol 1 1.73 2.08 1.33 1.34 0.84 1.540.88 1.05 1.11 0.87 Campesterol 1 0.74 1.13 0.47 0.60 0.76 0.75 0.830.90 1.20 1.21 Stigmasterol 1 0.45 1.00 0.34 0.30 0.50 0.55 0.65 0.851.42 1.27 Sitosterol 1 0.84 0.59 0.69 0.92 0.86 0.93 1.01 0.76 0.83 0.84*Highlighted entries indicate transgenic tobacco lines having areduction in sterols

Burley tobacco will be transformed with the HMG-CoA reductase cassetteusing Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for sterol content. It isexpected that approximately 50% of the lines tested will havesignificantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Burley tobacco that willbe created using the construct above will have significantly reducedlevels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the HMG-CoA reductaseinhibition cassette using Agrobacterium-mediated, Transbacter-mediated,or biolistic transformation and independent lines will be selected,regenerated, and transplanted in the greenhouse. Most of the independentlines grown in the greenhouse will be harvested and tested for sterolcontent. It is expected that approximately 50% of the lines tested willhave significantly less sterol than the parent strain of tobacco.Accordingly, it is expected that the transgenic Oriental tobacco thatwill be created using the construct above will have significantlyreduced levels of sterol and will generate significantly less PAHs uponpyrolysis, as compared to a conventional tobacco, a reference tobacco,or the parental strain of tobacco prior to genetic modification.

More embodiments concern still another RNAi construct designed to reducethe amount of a sterol in tobacco and thereby reduce production of a PAHupon pyrolysis of said transgenic tobacco. A fifth sterol-reducing RNAiconstruct has a developmentally regulated SMT2 inhibition cassette (SEQ.ID. No. 32). The developmentally regulated SMT2 inhibition cassettecomprises a cinnamyl alcohol dehydrogenase promoter (Bp 1-995) operablylinked to an antisense SMT2 nucleic acid (Bp 996-1775), which is joinedto a PAP 1 intron (Bp 1776-2955), which is joined to a sense nucleicacid of the SMT2 gene (Bp 2956-3735), which is joined to the RuBisCosmall subunit terminator (Bp 3736-4286). Accordingly, tobacco products(e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobaccoseeds, in barley, Flue-cured or Oriental comprising this RNAi constructare embodiments provided herein.

Flue-cured tobacco will be transformed with the developmentallyregulated SMT2 inhibition cassette using Agrobacterium-mediated,Transbacter-mediated, or biolistic transformation and independent lineswill be selected, regenerated, and transplanted in the greenhouse. Mostof the independent lines grown in the greenhouse will be harvested andtested for sterol content. It is expected that approximately 50% of thelines tested will have significantly less sterol than the parent strainof tobacco. Accordingly, it is expected that the transgenic Flue-curedtobacco that will be created using the construct above will havesignificantly reduced levels of sterol and will generate significantlyless PAHs upon pyrolysis, as compared to a conventional tobacco, areference tobacco, or the parental strain of tobacco prior to geneticmodification.

Burley tobacco will be transformed with the developmentally regulatedSMT2 inhibition cassette using Agrobacterium-mediated,Transbacter-mediated, or biolistic transformation and independent lineswill be selected, regenerated, and transplanted in the greenhouse. Mostof the independent lines grown in the greenhouse will be harvested andtested for sterol content. It is expected that approximately 50% of thelines tested will have significantly less sterol than the parent strainof tobacco. Accordingly, it is expected that the transgenic Burleytobacco that will be created using the construct above will havesignificantly reduced levels of sterol and will generate significantlyless PAHs upon pyrolysis, as compared to a conventional tobacco, areference tobacco, or the parental strain of tobacco prior to geneticmodification.

Oriental tobacco will be transformed with the developmentally regulatedSMT2 inhibition cassette using Agrobacterium-mediated,Transbacter-mediated, or biolistic transformation and independent lineswill be selected, regenerated, and transplanted in the greenhouse. Mostof the independent lines grown in the greenhouse will be harvested andtested for sterol content. It is expected that approximately 50% of thelines tested will have significantly less sterol than the parent strainof tobacco. Accordingly, it is expected that the transgenic Orientaltobacco that will be created using the construct above will havesignificantly reduced levels of sterol and will generate significantlyless PAHs upon pyrolysis, as compared to a conventional tobacco, areference tobacco, or the parental strain of tobacco prior to geneticmodification.

FIG. 6 illustrates a double-knock-out RNAi construct that can be used tocreate a reduced nicotine, TSNA, sterol transgenic tobacco thatgenerates a reduced amount of PAH upon pyrolysis. This double-knock-outRNAi construct has a A622/SMT2 inhibition cassette (SEQ. ID. No. 33) anda norflurazone selection cassette (SEQ. ID. No. 35). Starting from theright border (RB), the A622/SMT2 inhibition cassette comprises an RD2promoter (Bp 1-2010) operably linked to a A622 antisense nucleic acid(628 bp) (Bp 2011-2638) of a A622 gene, which is joined to a FAD2 intron(Bp 2639-3769), which is joined to a sense nucleic acid of the A622 gene(628 bp) (Bp3770-4397), which is joined to the GAD2 terminator (Bp4398-4670); which is joined to a cinnamyl alcohol dehydrogenase promoter(Bp 4671-5665) operably linked to an antisense SMT2 nucleic acid (Bp5666-6445), which is joined to a PAP 1 intron (Bp 6446-7625), which isjoined to a sense nucleic acid of the SMT2 gene (Bp 7626-8405), which isjoined to the RuBisCo small subunit terminator (Bp 8406-8956).Accordingly, tobacco products (e.g., cigarettes), tobacco, tobaccoplants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Orientalcomprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the construct shown in FIG.6 using Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid and sterolcontent. It is expected that approximately 50% of the lines tested willhave less than 1,000 ppm total alkaloid and a reduced amount of sterols,as compared to the parental strain of tobacco, and approximately 10% ofthe lines tested will have less than 500 ppm total alkaloid and areduced amount of sterols, as compared to the parental strain oftobacco. Accordingly, it is expected that the transgenic Flue-curedtobacco that will be created using the construct shown in FIG. 6 willhave significantly reduced levels of nicotine, TSNA, sterol, and willgenerate significantly less PAHs upon pyrolysis, as compared to aconventional tobacco, a reference tobacco, or the parental strain oftobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 6using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid and sterolcontent. It is expected that approximately 50% of the lines tested willhave less than 1,000 ppm total alkaloid and a reduced amount of sterols,as compared to the parental strain of tobacco, and approximately 10% ofthe lines tested will have less than 500 ppm total alkaloid and areduced amount of sterols, as compared to the parental strain oftobacco. Accordingly, it is expected that the transgenic Burley tobaccothat will be created using the construct shown in FIG. 6 will havesignificantly reduced levels of nicotine, TSNA, sterol, and willgenerate significantly less PAHs upon pyrolysis, as compared to aconventional tobacco, a reference tobacco, or the parental strain oftobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 6using Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid and sterolcontent. It is expected that approximately 50% of the lines tested willhave less than 1,000 ppm total alkaloid and a reduced amount of sterols,as compared to the parental strain of tobacco, and approximately 10% ofthe lines tested will have less than 500 ppm total alkaloid and areduced amount of sterols, as compared to the parental strain oftobacco. Accordingly, it is expected that the transgenic Orientaltobacco that will be created using the construct shown in FIG. 6 willhave significantly reduced levels of nicotine, TSNA, sterol, and willgenerate significantly less PAHs upon pyrolysis, as compared to aconventional tobacco, a reference tobacco, or the parental strain oftobacco prior to genetic modification.

FIG. 7 shows another RNAi construct that was used to generate severallines of reduced nicotine and TSNA tobacco. This RNAi construct has aQTPase inhibition cassette (SEQ. ID. No. 42) and a norflurazoneselection cassette (SEQ. ID. No. 35). Starting from the right border(RB), the QPTase inhibition cassette comprises an RD2 promoter (Bp1-2010) operably linked to an antisense fragment (360 bp) (Bp 2011-2370)of the QTPase gene, joined to a FAD2 intron (Bp 2371-3501), which isjoined to a sense fragment of the QTPase gene (360 bp) (Bp 3502-3861),which is joined to the nopaline synthase (NOS) terminator (Bp3862-4115). The selection cassette comprises the Actin 2 promoter (Bp1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp1162-2890) joined to the GapC terminator (Bp 2891-3387) at the leftborder (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco,tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured orOriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the construct shown in FIG.7 using Agrobacterium-mediated, Transbacter-mediated, or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid, as compared to the parental strain of tobacco,and approximately 10% of the lines tested will have less than 500 ppmtotal alkaloid, as compared to the parental strain of tobacco.Accordingly, it is expected that the transgenic Flue-cured tobacco thatwill be created using the construct shown in FIG. 7 will havesignificantly reduced levels of nicotine and TSNA, as compared to aconventional tobacco, a reference tobacco, or the parental strain oftobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 7using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid, as compared to the parental strain of tobacco,and approximately 10% of the lines tested will have less than 500 ppmtotal alkaloid, as compared to the parental strain of tobacco.Accordingly, it is expected that the transgenic Burley tobacco that willbe created using the construct shown in FIG. 7 will have significantlyreduced levels of nicotine and TSNA, as compared to a conventionaltobacco, a reference tobacco, or the parental strain of tobacco prior togenetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 7using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Oriental tobacco that will be created using the constructshown in FIG. 7 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

FIG. 8 shows another RNAi construct that was used to generate severallines of reduced nicotine and TSNA tobacco. This RNAi construct has aQTPase inhibition cassette (SEQ. ID. No. 43) and a norflurazoneselection cassette (SEQ. ID. No. 35). Starting from the right border(RB), the QPTase inhibition cassette comprises a PMTase1 promoter (Bp1-711) operably linked to an antisense fragment (360 bp) (Bp 712-1071)of the QTPase gene, joined to a FAD2 intron (Bp 1072-2202), which isjoined to a sense fragment of the QTPase gene (360 bp) (Bp 2203-2562),which is joined to the Gad2 terminator (Bp 2563-2835). The selectioncassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to amutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to theGapC terminator (Bp 2891-3387) at the left border (LB). Accordingly,tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobaccocells, tobacco seeds, in Burley, Flue-cured or Oriental comprising thisRNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 8using Agrobacterium-mediated transformation and more than about 98% ofputative transformants were successfully transformed. Of the independentlines, 200 plants were regenerated, transplanted in the greenhouse,harvested and tested for alkaloid content. A total of 75 lines wereidentified as having less than 1,000 ppm total alkaloid and no lineswere identified as having less than 500 ppm total alkaloid. Accordingly,the transgenic Flue-cured tobacco created using the construct shown inFIG. 8 has significantly reduced levels of nicotine and TSNA, ascompared to a conventional tobacco, a reference tobacco, or the parentalstrain of tobacco prior to genetic modification.

Burley tobacco was also transformed with the construct shown in FIG. 8using Agrobacterium-mediated transformation and more than about 98% ofputative transformants were successfully transformed. Of the independentlines, 201 plants were regenerated, transplanted in the greenhouse,harvested and tested for alkaloid content. A total of 86 lines wereidentified as having less than 3,000 ppm total alkaloid and 12 lineswere identified as having less than 1,000 ppm total alkaloid.Accordingly, it is understood by those skilled in the art that thetransgenic Burley tobacco created using the construct shown in FIG. 8also has significantly reduced levels of nicotine and TSNA, as comparedto a conventional tobacco, a reference tobacco, or the parental strainof tobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 8using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Oriental tobacco that will be created using the constructshown in FIG. 8 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

FIG. 9 shows another RNAi construct that was used to generate severallines of reduced nicotine and TSNA tobacco. This RNAi construct has aPMTase inhibition cassette (SEQ. ID. No. 44) and a norflurazoneselection cassette (SEQ. ID. No. 35). Starting from the right border(RB), the PMTase inhibition cassette comprises a truncated RD2 promoter(Bp 1-1061) operably linked to an antisense fragment (202 bp) (Bp1062-1263) of the PMTase gene, joined to an Act11 intron (Bp 1264-1418),which is joined to a sense fragment of the PMTase gene (262 bp) (Bp1419-1620), which is joined to the Gad2 terminator (Bp 1621-1893). Theselection cassette comprises the Actin 2 promoter (Bp 1-1161) operablylinked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890)joined to the GapC terminator (Bp 2891-3387) at the left border (LB).Accordingly, tobacco products (e.g., cigarettes), tobacco, tobaccoplants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Orientalcomprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 9using Agrobacterium-mediated transformation and more than about 98% ofputative transformants were successfully transformed. Of the independentlines, 100 plants were regenerated, transplanted in the greenhouse,harvested and tested for alkaloid content. A total of 86 lines wereidentified as having less than 1,000 ppm total alkaloid and 12 lineswere identified as having less than 500 ppm total alkaloid. Accordingly,the transgenic Flue-cured tobacco created using the construct shown inFIG. 9 has significantly reduced levels of nicotine and TSNA, ascompared to a conventional tobacco, a reference tobacco, or the parentalstrain of tobacco prior to genetic modification.

Burley tobacco was also transformed with the construct shown in FIG. 9using Agrobacterium-mediated transformation and more than about 98% ofputative transformants were successfully transformed. Of the independentlines, 99 plants were regenerated, transplanted in the greenhouse,harvested and tested for alkaloid content. A total of 29 lines wereidentified as having less than 3,000 ppm total alkaloid and no lineswere identified as having less than 1,000 ppm total alkaloid.Accordingly, it is understood by those skilled in the art that thetransgenic Burley tobacco created using the construct shown in FIG. 9also has significantly reduced levels of nicotine and TSNA, as comparedto a conventional tobacco, a reference tobacco, or the parental strainof tobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 9using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Oriental tobacco that will be created using the constructshown in FIG. 9 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

FIG. 10 shows another RNAi construct that was used to generate severallines of reduced nicotine and TSNA tobacco. This RNAi construct has aPMTase inhibition cassette (SEQ. ID. No. 45) and a norflurazoneselection cassette (SEQ. ID. No. 35). Starting from the right border(RB), the PMTase inhibition cassette comprises a RD2 promoter (Bp1-2006) operably linked to an antisense fragment (344 bp) (Bp 2007-2350)of the PMTase gene, joined to an Fad2 intron (Bp 2351-3481), which isjoined to a sense fragment of the PMTase gene (344 bp) (Bp 3482-3825),which is joined to the Gad2 terminator (Bp 3826-4098) at the left border(LB). The selection cassette comprises the Actin 2 promoter (Bp 1-1161)operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp1162-2890) joined to the GapC terminator (Bp 2891-3387) at the leftborder (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco,tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured orOriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 10using Agrobacterium-mediated transformation and more than about 98% ofputative transformants were successfully transformed. Of the independentlines, 66 plants were regenerated, transplanted in the greenhouse,harvested and tested for alkaloid content. A total of 44 lines wereidentified as having less than 1,000 ppm total alkaloid and 17 lineswere identified as having less than 500 ppm total alkaloid. Accordingly,the transgenic Flue-cured tobacco created using the construct shown inFIG. 10 has significantly reduced levels of nicotine and TSNA, ascompared to a conventional tobacco, a reference tobacco, or the parentalstrain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 10using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid, as compared to the parental strain of tobacco,and approximately 10% of the lines tested will have less than 500 ppmtotal alkaloid, as compared to the parental strain of tobacco.Accordingly, it is expected that the transgenic Burley tobacco that willbe created using the construct shown in FIG. 10 will have significantlyreduced levels of nicotine and TSNA, as compared to a conventionaltobacco, a reference tobacco, or the parental strain of tobacco prior togenetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 10using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid and approximately 10% of the lines tested willhave less than 500 ppm total alkaloid. Accordingly, it is expected thatthe transgenic Oriental tobacco that will be created using the constructshown in FIG. 10 will have significantly reduced levels of nicotine andTSNA, as compared to a conventional tobacco, a reference tobacco, or theparental strain of tobacco prior to genetic modification.

FIG. 11 shows another RNAi construct that was used to generate severallines of reduced nicotine and TSNA tobacco. This RNAi construct (SEQ IDNo 49) has a QTPase inhibition cassette (SEQ. ID. No. 42) and akanamycin selection cassette (SEQ. ID. No. 48). Starting from the rightborder (RB), The QPTase inhibition cassette comprises an RD2 promoter(Bp 1-2010) operably linked to an antisense fragment (360 bp) (Bp2011-2370) of the QTPase gene, joined to a FAD2 intron (Bp 2371-3501),which is joined to a sense fragment of the QPTase gene (360 bp) (Bp3502-3861), which is joined to the NOS terminator (Bp 3862-4115). Theselection cassette comprises the nopaline synthase (NOS) promoter (Bp4116-4422) operably linked to a neomycin phosphotransferase (NPTII) gene(Bp 4435-5229) joined to the NOS terminator (Bp 5619-5872) at the leftborder (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco,tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured orOriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 11using Agrobacterium-mediated transformation and more than about 98% ofputative transformants were successfully transformed. Of the independentlines, 99 plants were regenerated, transplanted in the greenhouse,harvested and tested for alkaloid content. A total of 43 lines wereidentified as having less than 1,000 ppm total alkaloid and 15 lineswere identified as having less than 500 ppm total alkaloid. Accordingly,the transgenic Flue-cured tobacco created using the construct shown inFIG. 11 has significantly reduced levels of nicotine and TSNA, ascompared to a conventional tobacco, a reference tobacco, or the parentalstrain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 11using Agrobacterium-mediated, Transbacter-mediated or biolistictransformation and independent lines will be selected, regenerated, andtransplanted in the greenhouse. Most of the independent lines grown inthe greenhouse will be harvested and tested for alkaloid content. It isexpected that approximately 50% of the lines tested will have less than1,000 ppm total alkaloid, as compared to the parental strain of tobacco,and approximately 10% of the lines tested will have less than 500 ppmtotal alkaloid, as compared to the parental strain of tobacco.Accordingly, it is expected that the transgenic Burley tobacco that willbe created using the construct shown in FIG. 11 will have significantlyreduced levels of nicotine and TSNA, as compared to a conventionaltobacco, a reference tobacco, or the parental strain of tobacco prior togenetic modification.

Oriental tobacco was transformed with the construct shown in FIG. 11using Agrobacterium-mediated transformation and more than about 98% ofputative transformants were successfully transformed. Of the independentlines, 122 plants were regenerated, transplanted in the greenhouse,harvested and tested for alkaloid content. A total of 22 lines wereidentified as having less than 1,000 ppm total alkaloid and 6 lines wereidentified as having less than 500 ppm total alkaloid. Accordingly, thetransgenic Flue-cured tobacco created using the construct shown in FIG.11 has significantly reduced levels of nicotine and TSNA, as compared toa conventional tobacco, a reference tobacco, or the parental strain oftobacco prior to genetic modification.

It should be emphasized that other promoters and terminators can be usedwith the nucleic acids provided herein interchangeably. Although RD2(SEQ. ID. No. 13, 37, or 50) is a preferred root-specific promoter,there are other root-specific promoters that can be used, as well. Forexample, the putrescene methyl transferase 1 promoter (PMT-1) (SEQ. ID.No. 14) is a root-specific promoter that can be used in place of the RD2promoter in any of the constructs described above. Similarly, althoughthe actin2 promoter (SEQ. ID. No. 16) is preferred for drivingexpression of a norflurazone resistance gene, other constitutivepromoters such as the GapC promoter (SEQ. ID. No. 15), the tobaccoalcohol dehydrogenase (ADP) (SEQ. ID. No. 17) and the Arabidopsisribosomal protein L2 (RPL2P) (SEQ. ID. No. 18) can be used to driveexpression of the norflurazone resistance gene. Additionally,developmentally regulated promoters such as, cinnamyl alcoholdehydrogenase (SEQ. ID. No. 19) and metallothionein I promoter (SEQ. ID.No. 20) can be used interchangeable with the cassettes described herein.

Further, in some embodiments, a plurality of constitutive promoters, intandem, can be used to drive expression of the norflurazone resistancegene. Additionally, a plurality of root-specific promoters can be usedto drive expression one or more of the inhibition cassettes describedabove (e.g., the QTPase inhibition cassette, the PMTase inhibitioncassette, the A622 inhibition cassette, a sterol inhibition cassette, ora double-knockout inhibition cassette). Developmentally regulatedpromoters, a plurality of developmentally regulated promoters,constitutive promoters, or a plurality of constitutive promoters canalso be used to drive expression of one or more of the inhibition orselection cassettes described above. Accordingly, any promoter operablein tobacco can be used to drive expression of any of the inhibitioncassettes or the selection cassette described herein (e.g., nos, 35S, orCAMV). Terminators, such as GAD2 terminator (SEQ. ID. No. 21), NOSterminator (SEQ ID No 38) and the FAD 2 (SEQ. ID. No. 22) or PAP1introns can be used interchangeably, as well.

Other embodiments provided herein concern the discovery of severalmutants of the phytoene desaturase gene that confer resistance to theherbicide norflurazone (e.g., SEQ. ID. Nos. 10, 11, and 12). Theseherbicide resistance genes were used as selectable markers in thetransformations above. Typically, the selection was accomplished byintroducing the transformed plant tissue to the norflurazone (e.g.,0.005 uM-0.1 uM conc.). That is, the concentration of norflurazone thatcan be used to select positive transformants containing a norflurazoneresistance gene, as described herein can be at least, less than, greaterthan, or equal to 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, or 1.0 uM. Preferably, less than or equal to 0.05 uMconcentration of norflurazone is used when selecting transformants withFlue-cured tobacco and less than or equal to 0.0125 uM concentrationnorflurazone is used when selecting transformants with Burley tobacco.As the plantlet develops, selection was accomplished by differentiatingthe green shoots (positive transformants) from the yellow or whiteshoots (negative transformants). Once selection was made, the herbicidewas removed and the plantlet was allowed to develop in the greenhouse.

The norflurazone resistant phytoene desaturase mutants (PDSM-1, PDSM-2,and PDSM-3) were generated by site-directed mutagenesis of particularregions of the gene believed to be involved in binding of the herbicide.Constructs carrying the various PDSM genes were then transferred totobacco leaf disks by conventional Agrobacterium transformation and theresistance to norflurazone was analyzed at various concentrations. Afterseveral iterations, the mutants described as SEQ. ID. Nos. 10, 11, and12, were identified as sequences that confer resistance to norflurazone.Accordingly, embodiments provided herein concern the PDSM genesdescribed herein, their use in plants as selectable markers to identifyplant cells that contain a transformed gene, whether in tissue cultureor in the field, and methods of identifying new PDSM genes that confernorflurazone resistance.

In a first selection construct, the Arabidopsis phytoene desaturase gene(PDS) (SEQ. ID. No. 36) was mutated using site-directed mutagenesis,such that a T to G mutation at position 1478, resulting in a Valine toGlycine change at amino acid residue 493 was created. To generate thenorflurazone resistance gene, the open reading frame of the Arabidopsisphytoene desaturase gene was amplified and cloned into the TOPO vector(Invitrogen). A single base pair change from T-G at nucleotide position1478, leading to a Valine to Glycine change at amino acid residue 493,was introduced using QuickChange Site-directed Mutagenisis Kit(Stratgene). The point mutation was verified by sequencing and theresultant mutant was named PDSM-1 (SEQ. ID. No. 10). The 1.729 Kb PDSM1sequence was then amplified and ligated into the binary vector pWJ001, apCambia derivative that contained the RNAi cassettes above, which wasthen introduced into Agrobacterium tumefaciens. A similar approach wasused to generate the PDSM-2 and PDSM-3 mutants described in the sequencelisting as SEQ. ID. NOs. 11 and 12.

That is, in a second selection construct, the Arabidopsis phytoenedesaturase gene (PDS) (SEQ. ID. No. 36) was mutated using site-directedmutagenesis, such that a G to C mutation at position 863, resulting in aArginine to Proline change at amino acid residue 288 was created. Togenerate the norflurazone resistance gene, the open reading frame of theArabidopsis phytoene desaturase gene was amplified and cloned into theTOPO vector (Invitrogen). A single base pair change was introduced usingQuickChange Site-directed Mutagenisis Kit (Stratgene). The pointmutation was verified by sequencing and the resultant mutant was namedPDSM-2. The 1.729 Kb PDSM-2 sequence was then amplified and ligated intothe binary vector pWJ001, a pCambia derivative that contained the RNAicassettes above, which was then introduced into Agrobacteriumtumefaciens

Further, in a third selection construct, the Arabidopsis phytoenedesaturase gene (PDS) (SEQ. ID. No. 36) was mutated using site-directedmutagenesis, such that a T to C mutation at position 1226, resulting ina Leucine to Proline change at amino acid residue 409 was created. Togenerate the norflurazone resistance gene, the open reading frame of theArabidopsis phytoene desaturase gene was amplified and cloned into theTOPO vector (Invitrogen). A single base pair change was introduced usingQuickChange Site-directed Mutagenisis Kit (Stratgene). The pointmutation was verified by sequencing and the resultant mutant was namedPDSM-3. The 1.729 Kb PDSM-2 sequence was then amplified and ligated intothe binary vector pWJ001, a pCambia derivative that contained the RNAicassettes above, which was then introduced into Agrobacteriumtumefaciens

Accordingly, embodiments provided herein concern methods of identifyinga mutation on a phytoene desaturase gene that confers resistance to anherbicide, preferably norflurazone. By one approach, a phytoenedesaturase gene is provided, preferably SEQ. ID. No. 36, a nucleotide insaid gene is mutated so as to generate a mutant phytoene desaturasegene, said mutant phytoene desaturase gene is transformed to a plantcell so as to generate a plant cell comprising said mutant phytoenedesaturase gene, said plant cell comprising said mutant phytoenedesaturase gene is then contacted with an herbicide, preferablynorflurazone, and the presence or absence of a resistance to saidherbicide is identified, whereby the presence of a resistance to saidherbicide identifies said mutation as one that confers resistance tosaid herbicide. By one approach, the entire sequence of a phytoenedesaturase gene (e.g., SEQ. ID. NO. 36) is mutated one residue at a timeand each mutant is screened for resistance to the herbicide.Accordingly, embodiments provided herein include compositions (e.g.,nucleic acid constructs or cassettes, plant cells, plants, tobacco, ortobacco products) that comprise, consist, consist essentially of amutant phytoene desaturase nucleic acid of SEQ. ID. NO. 10, 11, or 12 orfragment thereof at least or equal to 30, 50, 100, 200, 400, 500, 700,900, 1000, 1200, 1400, 1600, or 1700 consecutive nucleotides of inlength that confers resistance to an herbicide, in particularnorflurazone. Embodiments provided herein also include compositions(e.g., nucleic acid constructs or cassettes, plant cells, plants,tobacco, or tobacco products) comprising the mutant phytoene desaturaseprotein or fragments thereof (e.g., at least 15, 25, 50, 100, 200, 300,400, 500 consecutive amino acids of a protein encoded by SEQ. ID. Nos.10, 11, or 12) that confer resistance to an herbicide, in particularnorflurazone.

The nucleic acid sequences, cassettes, and constructs described hereincan also be altered by mutation such as substitutions, additions, ordeletions that provide for sequences encoding functionally equivalentmolecules. Due to the degeneracy of nucleotide coding sequences, otherDNA sequences that encode substantially the same amino acid sequence canbe used in some embodiments provided herein. These include, but are notlimited to, nucleic acid sequences comprising all or portions of thenucleic acid embodiments described herein that complement said sequencesand have been altered by the substitution of different codons thatencode a functionally equivalent amino acid residue within the sequence,thus producing a silent change. In some contexts, the phrase“substantial sequence similarity” in the present specification andclaims means that DNA, RNA or amino acid sequences which have slight andnon-consequential sequence variations from the actual sequencesdisclosed and claimed herein are considered to be equivalent to thesequences provided herein. In this regard, “slight and non-consequentialsequence variations” mean that “similar” sequences (i.e., the sequencesthat have substantial sequence similarity with the DNA, RNA, or proteinsdisclosed and claimed herein) will be functionally equivalent to thesequences disclosed and claimed in the present invention. Functionallyequivalent sequences will function in substantially the same manner toproduce substantially the same compositions as the nucleic acid andamino acid compositions disclosed and claimed herein.

Additional nucleic acid embodiments include sequences that are at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, and 100% identical to the nucleic acids,nucleic acid constructs, and nucleic acid cassettes provided herein.Preferably these sequences also perform the functions of the particularnucleic acid embodiment (e.g., inhibition of nicotine, nornicotine, orsterol production or confer resistance to norflurazone). Determinationsof sequence similarity are made with the two sequences aligned formaximum matching; gaps in either of the two sequences being matched areallowed in maximizing matching. Gap lengths of 10 or less are preferred,gap lengths of 5 or less are more preferred, and gap lengths of 2 orless still more preferred.

Additional nucleic acid embodiments also include nucleic acids thathybridize to the nucleic acid sequences disclosed herein under low,medium, and high stringency, wherein said additional nucleic acidembodiments also perform the function of the particular embodiment(e.g., inhibit nicotine, nornicotine, or sterol production or conferresistance to norflurazone). Identification of nucleic acids thathybridize to the embodiments described herein can be determined in aroutine manner. (See J. Sambrook et al., Molecular Cloning, A LaboratoryManual (2d Ed. 1989) (Cold Spring Harbor Laboratory)). For example,hybridization of such sequences may be carried out under conditions ofreduced stringency or even stringent conditions (e.g., conditionsrepresented by a wash stringency of 0.3 M NaCl, 0.03 M sodium citrate,0.1% SDS at 60 degrees C., or even 70 degrees C.). Preferably thesesequences also perform the functions of the particular nucleic acidembodiment (e.g., inhibition of nicotine, nornicotine, or sterolproduction or confer resistance to norflurazone).

Accordingly embodiments provided herein also include compositionscomprising, consisting of, or consisting essentially of: (a) the nucleicacid sequences shown in the sequence listing (SEQ. ID. NOS. 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50); (b) nucleotide sequences encoding theamino acid sequences encoded by the nucleic acids of the sequencelisting (SEQ. ID. NOS. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50);(c) any nucleotide sequences that hybridizes to the complement of thesequences shown in the sequence listing (SEQ. ID. NOS. 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49 or 50) under stringent conditions, e.g.,hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7.0% sodium dodecylsulfate (SDS), 1 mM EDTA at 50 degrees C. and washing in0.2.times.SSC/0.2% SDS at 50 degrees C.; and (d) any nucleotide sequencethat hybridizes to the complement of the sequences shown in the sequencelisting (SEQ. ID. NOS. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50)under less stringent conditions (e.g., hybridization in 0.5 M NaHPO₄,7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 37 degrees C. andwashing in 0.2.times.SSC/0.2% SDS at 37 degrees C. Preferably thesesequences also perform the functions of the particular nucleic acidembodiment (e.g., inhibition of nicotine, nornicotine, or sterolproduction or confer resistance to norflurazone). Embodiments providedherein also include peptides encoded by the nucleic acid sequences of(a), (b), (c), or (d), above.

The examples described herein demonstrate that several different RNAiconstructs can be used to effectively reduce the levels of nicotine,nornicotine, and sterols in tobacco. Additionally, these examplesdemonstrate that several mutant phytoene desaturase genes, which conferresistance to the herbicide norflurazone, have been created and thatselection cassettes comprising these herbicide resistant nucleic acidscan be used to determine the presence of a linked gene in transformedtobacco cells. Additionally, the norflurazone resistance nucleic acidsdescribed herein can be used in a general sense (e.g., in plants otherthan tobacco) to efficiently select positively transformed plant cellsfrom plant cells that do not contain a construct comprising thenorflurazone resistance gene. Thus, the norflurazone selection cassetteor the norflurazone resistance gene described herein can be used toconfer resistance to norflurazone in plants including, but not limitedto, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.),alfalfa (Medicago saliva), rice (Orya sativa), rape (Brassica napus),rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare),sunflower (Helianthus annus), wheat (Triticum aestivum), soybean(Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum),peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato(Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.),coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees(Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana(Musa spp.), avocado (Persea americana), fig (Ficus casica), guava(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),papaya (Carica papaya), cashew (Anacardium occidentale), macadamia(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Betavulgaris), apple (Malus pumila), blackberry (Rubus), strawberry(Fragaria), walnut (Juglans regia), grape (Vitis vinifera), apricot(Prunus armeniaca), cherry (Prunus), peach (Prunus persica), plum(Prunus domestica), pear (Pyrus communis), watermelon (Citrullusvulgaris), duckweed (Lemna), oats, barley, vegetables, ornamentals,conifers, and turfgrasses (e.g., for ornamental, recreational or foragepurposes). Vegetables include Solanaceous species (e.g., tomatoes;Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), carrots(Caucuis carota), cauliflower (Brassica oleracea), celery (apiumgraveolens), eggplant (Solanum melongena), asparagus (Asparagusofficinalis), ochra (Abelmoschus esculentus), green beans (Phaseolusvulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.),members of the genus Cucurbita such as Hubbard squash (C. Hubbard),Butternut squash (C. moschtata), Zucchini (C. pepo), Crookneck squash(C. crookneck), C. argyrosperma, C. argyrosperma ssp, C. digitata, C.ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii, andmembers of the genus Cucumis such as cucumber (Cucumis sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentalplants include azalea (Rhododendron spp.), hydrangea (Macrophyllahydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips(Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherima),and chrysanthemum. Conifers, which may be employed in practicing thepresent invention, include, for example, pines such as loblolly pine(Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinusponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinusradiata), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true firs such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Turfgrass include but are not limited to zoysia grasses, bentgrasses, fescuegrasses, bluegrasses, St. Augustine grasses, Bermuda grasses, buffalograsses, ryegrasses, and orchard grasses. Also included are plants thatserve primarily as laboratory models, e.g., Arabidopsis. Preferredplants for use in the present methods include (but are not limited to)legumes, solanaceous species (e.g., tomatoes), leafy vegetables such aslettuce and cabbage, turf grasses, and crop plants (e.g., tobacco,wheat, sorghum, barley, rye, rice, corn, soybean, cotton, cassava, andthe like), and laboratory plants (e.g., Arabidopsis). While any plantmay be used to carry out this aspect provided herein, tobacco plants areparticularly preferred.

Further, embodiments provided herein concern the production ofnorflurazone-resistant or tolerant plants, which can be sprayed with theherbicide in the field. In this manner, weeds and non-transformed plantswill die after contact with the herbicide but plants containing theconstruct harboring the norflurazone resistance gene will survive. Inone embodiment, for example, a norflurazone-containing herbicide isapplied to the plant comprising the DNA constructs provided herein, andthe plants are evaluated for tolerance to the herbicide. Any formulationof norflurazone can be used for testing plants comprising the DNAconstructs provided herein. The testing parameters for an evaluation ofthe norflurazone tolerance of the plant will vary depending on a numberof factors. Factors would include, but are not limited to the type ofnorflurazone formulation, the concentration and amount of norflurazoneused in the formulation, the type of plant, the plant developmentalstage during the time of the application, environmental conditions, theapplication method, and the number of times a particular formulation isapplied. For example, plants can be tested in a greenhouse environmentusing a spray application method. The testing range using norflurazonecan include, but is not limited to 0.5 oz/acre to 500 oz/acre. That is,the amount of herbicide that can be applied to transgenic plantscontaining a norflurazone-resistance gene in a field can be less than,equal to, or more than 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400, or500 oz/acre. In some embodiments, the norflurazone application rate is2.24 kg to 4.48 kg ai/hectare (2 to 4 lbs ai/acre) or 2.8 to 5.6 kggranules/hectare (2.5 to 5 lb/acre) or 234 L/hectare (25 gal/acre) insolution. Higher amounts are preferred for finer textured soils or whenlonger residual activity is desired.

The preferred commercially effective range can be from 25 oz/acre to 100oz/acre of norflurazone, depending on the crop and stage of plantdevelopment. A crop can be sprayed with at least one application of anorflurazone. For testing in cotton an application of 32 oz/acre at the3-leaf stage may be followed by additional applications at later stagesin development. For wheat, corn, soybean, and tobacco an application of32 oz/acre of norflurazone at the 3-5 leaf stage can be used. The testparameters can be optimized for each crop in order to find theparticular plant comprising the constructs provided herein that confersthe desired commercially effective norflurazone tolerance level. Thesection below describes typical curing methods which may be used toprepare the tobacco once it is harvested.

a) Heterologous Expression

Tobacco has well-established transformation procedures andwell-characterized regulatory elements for the control of transgeneexpression. Tobacco also has a high biomass yields and rapidscalability, which makes it a very suitable platform for commercialmolecular farming. Since tobacco is a non-food and non-feed crop it alsocarries a reduced risk that the transgenic material or recombinantproteins would contaminate animal feed and would enter the human foodchain. Because conventional or wild-type tobacco has a high content ofnicotine and other toxic alkaloids, however, investigators have notexplored the ability to use tobacco as a bioreactor. Further, the highcost of nicotine removal has steered investigators away from thistechnology.

The present disclosure, however, provides several types of geneticallymodified tobacco that can be used as a platform into which genesencoding commercially valuable compounds can be introduced. That is, byusing genetically modified tobaccos having reduced levels of nicotine,sterols, and/or TSNAs, as bioreactors, it is contemplated that manycommercially valuable industrial oils, pharmaceuticals, dietarysupplements can be obtained with fewer processing steps (e.g., theremoval of nicotine is no longer required). Accordingly, someembodiments concern tobaccos that are genetically modified to have areduced level of nicotine, sterols, and/or TSNAs, further comprising aheterologous gene that produces a medicinal compound, industrial oil, ordietary supplement, which can be harvested and/or isolated or purifiedfrom said tobacco. Compounds generated in this manner can be used for avariety of applications such as the preparation of immunogens, vaccines,cooking oils, pharmaceuticals and dietary supplements. Techniques forthe production of medicinal compounds in low-nicotine tobacco, such as aprotein, for industrial or pharmaceutical application has been describedin the art. It is contemplated, that these techniques can be readilyused with the tobaccos and techniques that are described herein.

As an exemplary, non-liming example, the N-terminal fragment of SARS-CoVS protein (S1) can be expressed in low-nicotine tobacco plants, as isknown in the art and exemplified in Pogrebnyak et al., Proc. Natl. Acad.Sci. USA (2005) 102:9062-9067. Incorporation of the S1 fragment intoplant genomes as well as its transcription can be confirmed by PCR andRT-PCR analyses. High levels of expression of recombinant S1 protein canbe observed in several transgenic lines by Western blot analysis usingspecific antibodies. Mammals parenterally primed with tobacco-derived S1protein can have sera containing SARS-CoV-specific IgG as detected byWestern blot and ELISA analysis.

The original gene encoding the human SARS-CoV spike glycoprotein (strainTOR2, National Center for Biotechnology Information no. NC 004718) isknown in the art. DNA encoding a 79-kDa S protein fragment,corresponding to amino acids 14-714, can be amplified by two consecutiverounds of PCR to generate XbaI and SacI sites at the 5′ and 3′ ends,respectively, by using the following primers: SP-F1-CCT TGC GCT TCT CAGCCA CGC AAA CTC AAG AGG ATC GCA TCA CCA TCA CCA TCA CAG TGA CCT TGA CCGGTG CAC (SEQ ID NO 51), XbaI-F2-ATA ATC TAG ATG ATC ATG GCT TCC TCC AAGTTA CTC TCC CTA GCC CTC TTC CTT GCG CTT CTC AGC CAC G (SEQ ID NO 52),and SacI-HDELR-ATT CGA GCT CTT AAA GTT CAT CAT GAG CCA TAG AAA CAG GCATTA CT (SEQ ID NO 53). The expression cassette of SARS-CoV 51 proteincan contain the plant-derived 23-aa {MIMASSKLLSLALFLALLSHANS (SEQ. ID.No. 54}, signal peptide (SP), and a histidine tag {RGSHHHHHH (SEQ. ID.NO. 55} at the N-terminal portion of the resulting 79-kDa polypeptide.After addition of the plant-specific endoplasmic reticulum retentionsignal {HDEL (SEQ. ID. NO. 56}, the cassette can be subcloned into theXbaI/SacI site of the plant binary vector pE1801, which is known as asuper promoter, followed by a tomato etch virus translation enhancer.The vector also can contain the npt II gene for kanamycin selection oftransgenic plants. Plasmid pE1801-79SHDEL can be electroporated intoAgrobacterium tumefaciens strain LBA4404 and used for planttransformations.

A genetically modified reduced nicotine and reduced TSNA tobacco, madeas described herein, can be used as the platform. The low-nicotine/TSNAtobacco can be transformed by Agrobacterium-mediated transformation asis known in the art. Independent kanamycin-resistant (KmR) tobacco linescan be used for molecular analyses. The presence of the spike gene intransgenic plants can be confirmed by PCR using genomic DNA. KmRtransgenic plants with PCR-confirmed presence of the S transgene can befurther analyzed for gene-specific mRNA expression by quantitativeRT-PCR as is known in the art. Western blot analysis of transgenic lineswith polyclonal SARS-specific antibodies Sm and Sn can confirm thepresence of SARSCoV S-specific 79-kDa protein and its derivatives. KmRT1 tobacco lines (cv. LAMD-609) can be grown hydroponically to obtainlarge amounts of root tissue for immunological experiments. Western blotanalysis of T1 lines can reveal high levels of S protein expressioncomparable with the original T0 transgenic lines.

Immunological assessment of the plant-expressed S protein can beperformed in 6- to 8-week-old female BALB/c mice. For parenteralimmunization, mice can be injected three times at 2-week intervals withan equivalent of 50 mg of dry tobacco root material per mouse. Powderedplant material can be reconstituted with saline (1/1 by weight) justbefore immunization. First and second immunizations can be given s.c.with complete and incomplete Freund's adjuvant, respectively; the thirddose can be administered i.p. in saline. Sera can be collectedretroorbitally from each mouse before and 10 days after eachimmunization. Four weeks after the last immunization, mice can receivean i.p. booster dose of 1 μg of commercially obtained S peptide (CellSciences, Canton, Mass.) without adjuvant. After 10 days, mice can bekilled and exanguinated by heart puncture, and sera can be assayed byELISA and Western blot analysis. Solid-phase ELISA can be carried out asknown in the art MaxiSorp 96-well plates (Nalge Nunc) coated overnightat 4° C. with the same S peptides obtained from Cell Sciences at aconcentration of 1 μg/ml in PBS. Antigen-specific antibodies can bedetected by using the following antibodies: rabbit anti-mouse IgG(total) and anti-mouse IgG1 (both from BD Biosciences Pharmingen),anti-mouse IgG2a, IgG2b, IgG3, IgM, and IgA (all from Organon Teknika),and anti-mouse IgE (eBioscience, San Diego). A serum dilution with anOD450 of 0.15 units above background can be considered the ELISA titer.

III. Tobacco Products

Although the modified tobaccos described herein are preferably used tocreate tobacco products for human consumption (e.g., cigarettes, chew,snuff, plug, etc.) it should be realized that the tobacco describedherein can be used for other applications such as animal feed,pharmaceutical production, and, in particular, the gernation of proteins(e.g., antiviral or anti-oncogenic peptides or antibodies or fragmentsthereof). Preferably, however, the tobacco and methods provided hereincan be applied to any tobacco product, including, but not limited topipe, cigar and cigarette tobacco and chewing tobacco in any formincluding leaf tobacco, shredded tobacco or cut tobacco. The term“tobacco product” includes, but is not limited to, smoking materials(e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum,and lozenges. In numerous embodiments, the methods provided herein areapplied to tobacco used to create the tobacco product.

A. Reduced Risk Tobacco Products

Provided herein are reduced risk tobacco products. A reduced risktobacco product provided herein can be a traditionally configuredtobacco product containing a reduced risk tobacco, such as a modifiedtobacco as provided herein. A reduced risk tobacco product providedherein also can contain conventional tobacco and be configured to reducethe risk of using the tobacco product. An example of such a reduced risktobacco product is a cigarette containing a filter designed to reducethe risk associated with cigarette smoke. A reduced risk tobacco productalso can contain a reduced risk tobacco and be configured to reduce therisk of using the tobacco product. An example of such a reduced risktobacco product is a cigarette containing a reduced risk tobacco and afilter designed to reduce the risk associated with cigarette smoke. Insome such embodiments, the reduced risk tobacco and reduced riskconfiguration act synergistically to reduce the overall risk of usingthe tobacco product.

Typical configurations of a tobacco product that reduces the risk of thetobacco product will include a filter that reduces the risk of exposureto tobacco smoke. A filter can be configured to be used with any smokingtobacco product, including cigars, pipes and cigarettes, as is known inthe art. In one embodiment, the reduced risk tobacco product is acigarette containing a filter that reduces the risk of exposure totobacco smoke. Any of a variety of known filters that reduce the risk ofexposure to tobacco smoke can be used in the reduced risk tobaccoproducts provided herein, including, but not limited to, commerciallyavailable filters provided in cigarette products, and other filtersknown in the art, such as filters containing antioxidants, copper,carbon or activated charcoal, and/or paper-containing filters. Oneexemplary filter can be a filter containing an antioxidant or a radicalscavenger. Filters containing antioxidants or radical scavengers can beprepared according to known methods, as exemplified in U.S. Pat. Nos.6,832,612 and 6,415,798, herein expressly incorporated by reference intheir entireties. Another exemplary filter is a filter that can reducetobacco smoke-induced modulation of cell homeostasis, as can be assessedby determining, for example, modulation of the transcriptome orproteome, cell viability, or integrity of genetic material. Such filterscan include a compound that sequesters or intercepts harmful componentsthat generate DNA breaks, or enhance DNA breakage, thereby yielding afilter that removes harmful smoke components. For example, a filter cancontain flat aromatic compounds that can scavenge potential carcinogens(e.g., components of tar), where exemplary flat aromatic compoundsinclude caffeine and pontoxyfyllen. In some of the methods providedherein the filter comprises an interceptor of the carcinogen that hasaromatic chemical structure: the carcinogen associates then withinterceptor forming a complex that is retained in the filter.

The methods provided herein can be used for evaluating modifications totobacco product configurations. For example, the methods provided hereincan be used as assays for evaluating the effectiveness of a cigarettefiller or filter. These methods also can be used to test any effect thatcan be resultant from a particular tobacco product configuration incombination with a modified tobacco. For example, the methods providedherein can be used to test a selected filter in combination with amodified tobacco. Accordingly, the methods provided herein provide abasis for evaluating and developing a reduced risk tobacco product. Themethods provided herein further provide a basis for evaluating anyfurther reduction in risk that can result from specific combinations ofmodified tobaccos and filters.

As provided herein, methods can be used for testing modulation of cellhomeostasis by, for example, monitoring a molecular marker of modulationof cell homeostasis, when the cells are exposed to a tobacco compositionfrom a modified tobacco configured in a tobacco product with a filter,or configured in a plurality of tobacco products with a plurality ofdifferent filters. Similarly, methods can be used for testing modulationof cell homeostasis when the cells are exposed to a tobacco compositionfrom a plurality of modified tobaccos configured in a tobacco productwith a filter, or configured in a plurality of tobacco products with aplurality of different filters. Accordingly, a variety of combinationsof modified tobaccos and tobacco configurations can be tested for theirproperties of modulating cell homeostasis. The examples provided hereindemonstrate that the risk-reducing properties of a tobacco and therisk-reducing properties of a filter can be interrelated such that therisk-reducing properties of a particular filter can vary depending onthe type of tobacco used. The methods provided herein can be used toevaluate the degree to which a particular filter reduces the risk of oneor more tobacco products, and also can be used to evaluate the abilityof one or more filters to reduce the risk of a particular tobaccoproduct. The methods provided herein also can be used to evaluate theability of one or more filters in combination with one or more tobaccosto have additive risk-reducing properties, thereby forming an evenfurther reduced-risk tobacco product.

B. Methods for Evaluating Tobacco and/or Tobacco Product

Methods for Determining the Risk Potential of Tobacco and TobaccoProducts

Provided herein are several methods for identifying the propensity of atobacco or tobacco product to contribute to a tobacco related disease.Generally, these approaches are practiced by providing a tobacco,obtaining smoke or a smoke condensate from the tobacco, contacting acell with the smoke or smoke condensate, and identifying one or moreattributes of the contacted cell. Tobacco products contain a number ofcompounds that induce various types of changes to a cell, including celldamage, change in gene expression including mRNA and/or proteinexpression, mutations, chromosomal aberrations, aberrant sisterchromatid exchanges and micronuclei. Attributes of contacted cellsindicative of such tobacco-induced cell changes can be identified in themethods provided herein, which address changes in cell homeostasis, asindicated by changes in gene expression, genetic mutations oraberrations, and modulation of cell viability and/or apoptosis. Themethods provided herein can be used to determine affect of a tobacco ora tobacco product on a cell by determination of the presence, absence,or change in a molecular marker. For example, a molecular marker can bemonitored, which is indicative of an affect on mRNA, protein, DNAdamage, cell viability or apoptosis can be determined according to themethods provided herein or other methods generally known in the art,where monitoring of the molecular marker can be used to determine theaffect of a tobacco or tobacco product on cell homeostasis. Exemplaryaffects of a tobacco or tobacco product on a cell include, but are notlimited to, induction of a double-strand DNA break, inhibition ofapoptosis, inhibition of cell proliferation, and modulation of geneexpression, including modulation of the transcriptome and/or modulationof the proteome. Accordingly, the methods provided herein can be used toestablish a profile for a particular tobacco by employing assay methods,including assays that identify tobacco products that modulate cellhomeostasis from tobacco products that do not. For example, assays forinduction of damage of cellular genetic material or assays formodulation of gene expression can be used to differentiate reduced risktobacco products from conventional tobacco products. For example, themethods provided herein can be used to characterize a tobacco by assaymethods including an assay for the induction of a double-strand DNAbreak, inhibition of apoptosis, inhibition of cell proliferation,modulation of transcription, or modulation of translation.

Several other assays have classically been used to analyze tobacco forthe risk of adverse health effects. Traditionally, the first manner oftesting consists of analysis of cigarette smoke for various componentsthat can relate to health effects associated with smoking. A secondmanner of testing includes testing cigarette smoke tar on living cells.One of these tests detects changes in the genetic material of bacteria.Another test uses mouse cells grown in Petri dishes to detect potentialcancer-causing activity. A third manner of testing seeks to determine ifpeople smoke the tested tobacco cigarettes differently than thecomparable brand or type currently on the market. If the way thecigarettes are smoked is different, then the other manners of testingcan be repeated with the smoking machines set to reflect the change insmoking behavior. A fourth manner of testing examines the response ofanimals to cigarette smoke or tar. One such type of test looks forinflammation in the lungs of mice in response to cigarette smoke. Asecond test looks for tumor formation in the upper respiratory tract ofhamsters exposed to smoke. A third test looks for the cancer producingability of cigarette smoke tar by applying the tar to the skin of mice.Each manner of testing can include comparing tobacco cigarettes and boththe effects of mainstream and sidestream smoke can be tested.

During smoking, both mainstream smoke (inhaled by the smoker) andsidestream smoke (mainly from the burning end of the cigarette) aregenerated. While mainstream and sidestream smoke are qualitativelysimilar the quantity of specific components differs between the two.Additionally, modifications to the cigarette can independently affectthe composition of sidestream and mainstream smoke. It is concluded,therefore, that testing of tobacco or cigarettes can be assayed for bothmainstream and sidestream smoke.

Epidemiology is not a practical approach for addressing the issue of thehealth effects of changes in a cigarette composition. Because people cansmoke cigarettes differently (ex. longer or faster puffs) it can beimportant to consider whether these changes affect smoke chemistry andtherefore toxicity. For example, a new, cigarette type can result in asmoker taking longer puffs, which can then change the smoke chemistryand toxicity.

Testing, however, can examine the effects on toxicity of a single designchange in a cigarette or can examine the effects of a set of designchanges compared to an unchanged control. Testing protocols can followeither a screening or a tradeoff approach. In the screening approach newdesigns can be subjected to a series of tests each with criteria forpassing or failing. Designs that fail are eliminated from furthertesting, while those passing are subjected to additional scrutiny. Inthe tradeoff approach the relative changes in each test would beassessed in light of other information about the particular design.

The FTC method describes: how cigarettes are to be prepared for smoking,the type of smoking machine to use, the way the smoking machine shouldbe operated, the method for collecting smoke products, and ways tomeasure moisture content, nicotine, carbon monoxide and tar. Typicallyin the methods provided herein, the FTC protocol for studies ofcigarette smoke chemistry and toxicity are used.

Toxicity of cigarette smoke is directly related to the composition ofthe smoke and the composition of smoke can be changed if the way thecigarettes are smoked is changed.

There are a variety of chemical analyses that can be done to aid in thedetermination of the change in toxicity of a tobacco. These relate tothe chemical composition of tobacco smoke. The following lists thechemical composition analysis and the health effect associated with thecomponent or property measured: Total Particulate Matter (TPM;carcinogen), pH (effect on nicotine toxicity), Redox Potential(influence toxicity of whole smoke), Carbon Monoxide (reduces ability ofblood to carry oxygen), Nitrogen Oxides (NOx; increases nitrosamineformation, inhibits enzyme function), Hydrogen Cyanide (inhibits lungclearance, lowers ability of body to use oxygen), Hydrocarbons (benzene,butadiene; suspected or known carcinogens), Aldehydes (ex. formaldehyde,acrolein; inhibit lung clearance, animal carcinogens), Volatilenitrosamines (strong animal carcinogens), Tobacco-specific nitrosamines(strong animal carcinogens), Nicotine (associated with cardiovasculardisease), Phenols (enhance carcinogen action) Catechol (majorcarcinogen), and Polynuclear Aromatic Hydrocarbons (PNAs; major tumorinitiators).

There are also a variety of known cell toxicity tests that can beperformed in a relatively short time scale: bacterial mutagenicity test,animal cell test to detect potential carcinogens, and lung inflammationtest in animals. One test, the Ames test, uses certain types ofSalmonella bacteria to quantitatively assess the ability of a materialto cause mutations, such as mutations involved in the process ofcarcinogenesis. In this test a solution of collected smoke particulatesis mixed with the bacteria. Bacteria with the ability to grow in theabsence of a particular nutrient are scored as mutants.

The potential cancer-causing ability of chemicals can also be evaluatedusing a cell transformation assay. In this assay, solutions of smokeparticulates are given to animal cells grown in Petri dishes in thelaboratory. After several weeks the cells are examined under themicroscope. At this time the cells are scored for abnormal growthpatterns. The number of clusters of abnormally growing cells is thencompared among cigarette types.

In animal studies, inflammation of the lungs can be assessed. Thechanges measured in this test can be related to the development ofchronic obstructive pulmonary disease. In these tests mice can beexposed to whole smoke two times per day, for any number of daysaccording to the experimental design. At the end of the exposure periodthe animals would be killed and their lungs washed out to collectinflammatory cells. The numbers and kinds of the cells would bemeasured.

Two long-term animal tests for cancer causing ability of tobacco can beperformed. In the first, test cigarette tar is applied to the back skinof mice. Skin tumors are then scored over the life of the animals. Theuse of this test is based on two observations: (1) in studies of tumorformation by smoke in hamsters whole smoke is active but smoke free ofparticulates is not and (2) a large number of known carcinogens arecontained in the particulate portion of cigarette smoke.

The second test examines the tumor forming ability, of whole smoke inhamsters. A positive response can be observed in the larynx of hamstersexposed over their lifetime to whole cigarette smoke. In this test theanimals are exposed twice daily to the diluted smoke of one cigaretteevery day for their entire lives. Tumor formation is the endpointmeasured in this assay. Because the test is so labor intensive it isrecommended only as a last step in a series of tests.

These known methods for assaying tobacco toxicity have limitations interms of time length and/or expense relative to the assay methodsprovided herein. Accordingly, there is a long felt need for more rapidand less costly methods of analysis of tobacco products of differentcompositions. Despite the inefficiencies of the approaches above, it iscontemplated herein that these methods for assaying tobacco toxicity canbe used alone or in conjunction with the methods provided herein so asto provide additional information regarding the properties of thetobacco being characterized.

In the methods provided herein, one or more cells can be contacted witha tobacco composition such as tobacco smoke (TS), a tobacco smokecondensate (TSC), or total particulate matter (TPM), where exemplary TSand TSC are cigarette smoke (CS) and cigarette smoke condensate (CSC).Preparation of the tobacco composition used in the methods providedherein can be performed in accordance with the teachings herein and theknowledge and skill in the art. For example, TS can be collected using asmoking machine such as an INBIFO-Condor smoking machine, and TSC can becollected using cold traps, and TPM can be collected using a filter, asis known in the art. For example, CSC for testing can be prepared bypassing smoke through a series of cold traps containing glass beads uponwhich CSC condenses; the CSC can then be collected by washing the beadswith acetone as described in Mathewson, H. D. Beitrage zur Tabforschung.3(6):430-7. September 1966. In addition, cells can be contacted withsmoke provided in diluted form, where diluted smoke can be produced in adilution chamber, as known in the art. For example, a smoking setup cancontain a dilution chamber where the concentration of the smoke beingapplied to the cells can be varied by dilution with air in order toproduce different dosages and intensities of smoke. The dilution chambercan be located between the burning cigarette and the cell exposurechamber. In addition, cigarette particulate matter for testing can beprepared by passing smoke through a glass fiber filter which issubsequently washed with solvent to collect the sample as described inCoresta Recommended Method No. 23 (August 1991). Although thedescription herein provides several methods in the context ofcharacterizing tobacco and tobacco products that undergo pyrolysis(e.g., cigarettes, pipe tobacco, and cigars), similar approaches can beapplied to the evaluate snuff, chew, and other tobacco products that donot undergo pyrolysis. Accordingly, the methods provided herein are notlimited to smoke or smoke condensate, but can be applied to any tobaccocomposition known in the art. The preparation and analysis ofcompositions from such non-pyrolysis tobacco products is straightforwardgiven the teachings provided herein or otherwise known in the art.Methods for contacting cells with compositions from such non-pyrolysistobacco products also is straightforward given the teachings providedherein or otherwise known in the art.

The tobacco derived composition (i) can originate in a tobacco product,which can be either pure tobacco or a tobacco formulation (such as acigarette, cigar, pipe or chewing tobacco) having multiple compositionalelements, for example but not limited to structural elements, flavorchemicals and/or other additives, and (ii) can have multiple components(e.g., smoke or a smoke condensate, also referred to collectively as“smoke products”) or can be a single known or unidentified component(e.g., a single chemical compound). The composition can be “derived”from tobacco or a tobacco formulation (i) by simple physical separation;(ii) as a product of combustion or heating, (iii) by solvent extraction,(iv) by chemical reaction(s) or (v) by enzyme activity (e.g., smokeconcentrate treated with a microsomal cellular fraction or purifiedcytochrome P450).

In some methods provided herein, cells are contacted with TS, such asCS. The contacting of the cells with the CS, CSC, TS, TSC or TPM can beaccomplished using any method known to one of skill in the art,including but not limited to, placing said cells into a smoking machineor smoke chamber (e.g., CULTEX®) for a period of time to allow the cellsto be contacted with smoke, and/or providing a CSC or TSC to the mediafor a designated period of time (e.g., in 0.5% dimethylsulfoxide orother formulation). The contacting can be for any amount of time,however, preferably the cells are contacted for an amount of time thatdoes not result in nonviability of more than 50% of the cells. In someembodiments, the amount of time can be varied and the results arecompared. In a further embodiment, the cells are treated for an amountof time in which the gene expression is modulated, but the majority ofcells are still viable. That is, in some embodiments, the cells aretreated to a point in which the cells are at least, equal to, or morethan 1% viable, including but not limited to 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99%, and 100% viable.

In a another embodiment, the amount of time for contacting a cell withthe CS, CSC, TS, TSC or TPM is any amount selected from the groupconsisting of about at least, equal to, or more than 1 seconds to about24 hours, including but not limited to at least, equal to, or more than1 second, 15 seconds, 30 seconds, 45 seconds, 1 minute, 3 minutes, 5minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35minutes, 40 minutes, 45 minutes, 60 minutes, 1.5 hours, 2 hours, 2.5hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours,13 hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, 15.5 hours, 16hours, 16.5 hours, 17 hours, 17.5 hours, 18 hours, 18.5 hours, 19 hours,19.5 hours, 20 hours, 20.5 hours, 21 hours, 22 hours 23 hours and 24hours. In a further embodiment, the cells are contacted for less thanand including about 20 minutes. In yet another embodiment, the cells arecontacted for about 2 to about 20 minutes.

The amount of smoke with which the cells are contacted can be any of avariety of amounts according to the desired level of exposure. Forexample, smoke exposure can be performed in accordance with FTCparameters: 2.0 second puff duration, 35 mL puff every 60 seconds. Puffduration, volume and frequency can be increased or decreased to achievedifferent levels of smoke exposure, as desired. Similarly, smokecondensate or other tobacco compositions can be contacted with cells ata variety of different concentrations and for a variety of differentdurations, as desired. For example, smoke condensate at 20 mg/mL can becontacted with cells for any of the above-provided amounts of time, asdesired.

Tobacco smoke or smoke products can be treated prior to contacting thecells with the smoke or smoke product. For example, the smoke or smokeconcentrate can be contacted with a filter, such as a filter providedherein, for example by obtaining smoke or smoke condensate from acigarette after passing through a filter attached to the tobaccoproduct, such as a cigarette.

The cells suitable for use in the methods provided herein include humanas well as non-human cells, but are preferably human pulmonary cells(e.g., lung or bronchial cell), although cells of other systems impactedby smoking, including but not limited to cells of the upperaerodigestive tract (e.g., oral cavity including cheek, pharynx, larynx,and esophagus), bladder, stomach, kidney, pancreas, and blood (e.g.,lymphocytes, monocytes, neutrophils, esoinophils or basophils, orneoplastic blood cells such as myeloid leukemia cells); cells of thecardiovascular system (including endothelial cells, smooth muscle cells(e.g. from vessel walls, myocardial cells, etc.) and cells of the femalereproductive system (e.g. cells of the uterus, cervix, fallopian tubes,ovary, and placenta), can also be used. The cells can be normal or canbe neoplastic, metaplastic, dysplastic or malignant. The cells can becollected from a living organism (e.g., a pulmonary lavage specimen,tissue section such as a lung or bronchial section, oral mucosa sample,cheek swab, or sputum sample), can be primary cell cultures, or can beestablished cell cultures. In some embodiments, the cells can beobtained from a living organism, including a human, after the organismis contacted with a tobacco composition, for example, after a humanconsumes a cigarette. Cells collected from a living organism can becollected using any of a variety of known methods known in the art,according to the cell type to be collected (e.g., a cheek scrape or lunglavage). In specific, non-limiting embodiments, the cells can be NHBEcells, or can be human epithelian pulmonary type II cells, such as A549cells, or can be cells obtained from a human primary culture.

Many embodiments described herein employ NHBE cells that are maintainedin culture, and other embodiments employ human lung carcinoma cells(A549 cells). Although NHBE and A549 cells are preferred for the methodsdescribed herein, it should be understood that many other cells that aretypically contacted with tobacco or TS during the process of smoking(e.g., lung cells, bronchial cells, cells of the oral mucosa, pharynx,larynx, and tongue) can also be used. Additionally, many immortal celllines can be used with the methods described herein. Preferred cells foruse with the embodied approaches include, but are not limited to, humanbronchial cells (e.g., BEP2D or 16HBE140 cells), human bronchialepithelial cells (e.g., HBEC cells, 1198, or 1170-I cells), NHBE cells,BEAS cells (e.g., BEAS-2B), NCI-H292 cells, non-small cell lung cancer(NSCLC) cells or human alveolar cells (e.g., H460, H1792, SK-MES-1,Calu, H292, H157, H1944, H596, H522, A549, and H226), tongue cells(e.g., CAL 27), and mouth cells (e.g., Ueda-1)). Many of such culturesare available commercially or through a public repository (e.g., ATCC).Further, several techniques exist that allow for one to generate primarycultures of said cells and these primary cultures can be used with themethods described herein.

Conventional approaches in tissue culture can be used to establish andmaintain said cells in preparation for the methods described herein.That is, the cells may be grown in culture by any method known to one ofskill in the art and with the appropriate media and conditions. Thecells grown in culture may require feeder layers, for example. The cellsmay be grown to confluence or may be grown to less than confluencebefore, during, or after treatment. In some embodiments the cells aregrown to between about 10% and about 90% confluence, including but notlimited to, at least, equal to, or more than 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%confluence before contact with CS, CSC, TS, or TSC.

In some embodiments, the cells contacted and assayed in accordance withthe methods provided herein are manipulated to control and/or modify thepercentage of cells that are in one or more phases of the cell cycle.For example, the cells can be manipulated such that at least 50% of thecells of the population of cells are in the S phase. The cells usedherein can be manipulated to control the population of cells in one ormore of G0, G1, S, G2, or M phases of the cell cycle. For example, cellscan be manipulated such that at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, or 100%, of the population of cells are in G0, G1, S, G2, or Mphase. In another example, cells can be manipulated such that greaterthan 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the populationof cells are in G0, G1, S, G2, or M phase. In another example, cells canbe manipulated such that 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or100%, of the population of cells are in G0, G1, S, G2, or M phase. Thesection below describes several preferred methods for characterizingtobacco and tobacco products in greater detail.

Exemplary Assays

The methods provided herein for characterizing tobacco or a tobaccoproduct can be used in a variety of applications, including, but notlimited to, the comparison of two or more tobaccos or two or moretobacco products, identifying a modulation of cell homeostasis,identifying an induction of damage of cellular genetic material, oridentifying a modulation of gene expression including mRNA and/orprotein expression. The methods provided herein for characterizingtobacco also can be used for identifying a tobacco product that has areduced potential to contribute to a tobacco-related disease, and makinga tobacco product that has a reduced potential to contribute to atobacco-related disease. In addition to methods provided herein forcharacterizing tobacco or a tobacco product, additional methods known inthe art for characterizing tobacco or a tobacco product can be used inthe place of, or in conjunction with, the methods provided herein.

The methods of identifying a tobacco, identifying a compound in tobacco,identifying a tobacco product, and making a tobacco product providedherein, can in addition be utilized in methods of identifying two ormore tobaccos, identifying a compound in two or more tobaccos,identifying a tobacco product by comparing two or more tobacco products,and making a tobacco product by comparing two or more tobacco products.In some embodiments, the two or more tobaccos or tobacco products can becompared for their effect on cell homeostasis, gene expression includingmRNA and/or protein expression, or damage to genetic material of cells.In some embodiments, at least one tobacco or tobacco product can be areduced risk tobacco or tobacco product, respectively. In someembodiments, at least one tobacco can be a modified tobacco, such as achemically modified tobacco or a genetically modified tobacco. In someembodiments, at least one tobacco can be a reduced risk tobacco product,such as a tobacco product configured to contain a filter that reducesthe risk of tobacco exposure such as a filter provided herein. In oneexample, two or more tobaccos can be compared to identify a compound intobacco that modulates gene expression including mRNA and/or proteinexpression. In one example, two or more tobacco products can be comparedto identify a compound in tobacco that modulates gene expressionincluding mRNA and/or protein expression.

In some embodiments, a second tobacco product (e.g., a cigarette) iscompared to a first tobacco product (e.g., a cigarette) using themethods above so as to identify which of the two tobacco products isless likely to contribute to a tobacco-related disease. For example, afirst population of isolated human cells of the mouth, tongue, oralcavity, or lungs (e.g., NHBE cells), is contacted with a CS from a firsttobacco product (e.g., a “reduced risk full flavor” cigarette) in anamount and for a time sufficient to modulate cell homeostasis, such asinducing damage of cellular genetic material or modulating geneexpression including mRNA and/or protein expression. A second populationof isolated human cells of the mouth, tongue, oral cavity, or lungs(e.g., NHBE cells), preferably the same type of cell as used in theanalysis of the first tobacco product, is also contacted with a CS froma second tobacco product (e.g., a cigarette) in an amount and for thesame amount of time as used with the first product or for a timesufficient to modulate cell homeostasis by, for example, inducing damageof cellular genetic material or modulating gene expression includingmRNA and/or protein expression.

The data obtained from the analysis of the first tobacco product can becompared to the data obtained from the analysis of the second tobaccoproduct so as to identify, for example, an increased risk tobacco or acompound in tobacco. The data also can be used to identify a reducedrisk tobacco. The data further can be used to identify a tobacco productconfiguration, such as a filter, with increased risk or with reducedrisk. Thus, by analyzing the differences between the tobacco products,one can identify a tobacco product that has less potential to contributeto a tobacco related disease or to identify, for example, a firsttobacco product that has a reduced risk to contribute to atobacco-related disease, as compared to a second tobacco product or viceversa. By one technique, a tobacco product that is less likely tocontribute to a tobacco-related disease is identified because it causesless modulation of cell homeostasis. By another technique, a tobaccoproduct that is less likely to contribute to a tobacco-related diseaseis identified because it causes less modulation of cell homeostasisunder the same level of damage induced to cellular genetic material. Inanother technique, a tobacco product that is less likely to contributeto a tobacco-related disease is identified because it induces lessdamage to cellular genetic material. In another technique, a tobaccoproduct that is less likely to contribute to a tobacco-related diseaseis identified because it induces fewer or smaller in degree changes ingene expression such as changes in the transcriptome or changes in theproteome.

The methods provided herein can be used not only to identify a tobaccoproduct that has a reduced potential to contribute to a tobacco-relateddisease, as compared to a second tobacco product, but also to developtobacco products that have a reduced potential to contribute to atobacco-related disease, as compared to a second tobacco product. Forexample, by screening modified tobacco (e.g., chemically or geneticallymodified tobacco) or a tobacco product with a modified configuration inaccordance with the methods disclosed herein, one can rapidly determinewhether the modified tobacco or modified tobacco product has anincreased or decreased potential to contribute to a tobacco-relateddisease, as compared to the tobacco or tobacco product that is notmodified.

More embodiments concern methods to identify components of a tobaccoproduct that contribute to a tobacco-related disease, the selectiveremoval or inhibition of production of these components, and thedetermination that the removal of the component(s) results in a reducedrisk tobacco product. Such a determination that the removal of thecomponent(s) results in a reduced risk tobacco product can be indicatedby, for example, a molecular marker that is associated with atobacco-related disease. Exemplary molecular markers of atobacco-related disease include, but are not limited to a molecularmarker indicative of apoptosis, a molecular marker indicative ofdouble-stranded DNA breaks, an overexpressed or underexpressed mRNA, anoverexpressed or underexpressed polypeptide. In one example methods areprovided to identify components of a tobacco product that contribute toa tobacco-related disease, the selective removal or inhibition ofproduction of these components, and the determination that the removalof the component(s) modulates expression of a gene that is associatedwith a tobacco-related disease in a manner that reduces the potentialfor the tobacco product to contribute to a tobacco related disease. Itis contemplated that particular components of tobacco products are thefactors that modulate responses in human cells that contribute totobacco-related disease. It is further contemplated that modification ofthe tobacco product will, concomitantly, result in a modulation of theresponse in human cells contacted with the smoke from said modifiedtobacco product, which modulates the likelihood to contribute to atobacco-related disease relative to an unmodified tobacco product. Forexample, modification of genes that contribute to the production ofthese toxic components in tobacco (e.g., genetic engineering or chemicaltreatment) will, concomitantly, result in a modulation of the responsein human cells contacted with the smoke from said modified tobacco,which modulates the likelihood to contribute to a tobacco-relateddisease relative to tobacco prior to modification of thecomponent-producing gene. Accordingly, by selectively removing thecomponents from a tobacco product (e.g., by modifying a tobacco or atobacco product configuration) that induce the events that contribute totobacco-related disease in a human, one can develop tobacco productsthat are less likely to contribute to a tobacco-related disease.

Methods for Identifying a Tobacco or Tobacco Product that Modulates CellHomeostasis

Provided herein are methods for identifying a tobacco that modulatescell homeostasis by providing a tobacco, obtaining a tobacco compositionfrom the tobacco, contacting a cell with the tobacco composition, andidentifying the presence or absence of a modulation of cell homeostasisafter contact with the tobacco composition. In some embodiments, themethods provided herein can be used to identify a tobacco that affectscell homeostasis as can be monitored by, for example, determining thatthe tobacco induces double strand DNA breaks, modulates apoptosis,modulates cell proliferation, or induces expression of a gene that issilent during homeostasis or repress a gene that is active duringhomeostasis. In some embodiments, the tobacco composition can be smokeor smoke condensate. By modulation of homeostasis of a cell is meant thechange in the state of a cell upon contact of the cell by a tobacco ortobacco composition (e.g., tobacco smoke or tobacco smoke condensate),where the state of the cell can be the cell cycle, the apoptotic state,the expression levels of one or more genes as represented by mRNA levelsand/or protein levels or post-translational modifications to proteins.Typically modulation of cell homeostasis can be monitored by measurementof one or more molecular markers of the state of a cell. It iscontemplated herein that any tobacco-induced change in cell homeostasiscan serve as an indicator that said tobacco or tobacco composition maycontribute to a tobacco-related disease. Because it is known thattobacco or tobacco compositions can have a plurality of adverse affectson cells, it is contemplated that typically a less pronounced modulationof cell homeostasis after the cell is contacted with a tobacco ortobacco composition (e.g., tobacco smoke or tobacco smoke condensate),the greater propensity that the tobacco or tobacco composition willpresent a reduced level of risk for developing a tobacco related diseaserelative to a conventional or reference tobacco. Accordingly, it iscontemplated herein that a tobacco or tobacco composition characterizedas reduced risk tobacco or tobacco composition is one that, upon contactwith a cell, does not modulate cell homeostasis or modulates cellhomeostasis to a lesser degree than the cell homeostasis modulationinduced by a conventional or reference tobacco.

Also provided herein are methods of identifying a compound in tobaccothat modulates cell homeostasis by providing a first tobacco, obtaininga tobacco composition from the first tobacco, contacting a firstpopulation of cells with the tobacco composition from the first tobacco,identifying the degree of modulation of cell homeostasis in the firstpopulation of cells after contact with the tobacco composition from thefirst tobacco, providing a second tobacco that has been modified toreduce a compound in the second tobacco, obtaining a tobacco compositionfrom the second tobacco, contacting a second population of cells withthe tobacco composition from the second tobacco, and identifying thedegree of modulation of cell homeostasis after contact with the tobaccocomposition from the second tobacco, where an identification of areduction in the degree of modulation of cell homeostasis after contactwith the tobacco composition from the second tobacco identifies thecompound as one that modulates cell homeostasis. In some embodiments,the methods provided herein can be used to identify a compound intobacco that modulates apoptosis. In some embodiments, the methodsprovided herein can be used to identify a compound in tobacco thatmodulates cell proliferation. In some embodiments, the tobaccocomposition can be smoke or smoke condensate.

Also provided herein are methods of identifying a tobacco product thathas a reduced potential to contribute to a tobacco-related disease byproviding a first tobacco product, obtaining a tobacco composition fromthe first tobacco product, contacting a first population of cells withthe tobacco composition from the first tobacco product, identifying thedegree of modulation of cell homeostasis in the first population ofcells after contact with the tobacco composition from the first tobaccoproduct, providing a second tobacco product, obtaining a tobaccocomposition from the second tobacco product, contacting a secondpopulation of cells with the tobacco composition from the second tobaccoproduct, and identifying the degree of modulation of cell homeostasisafter contact with the tobacco composition from the second tobaccoproduct, where an identification of a reduction in the degree ofmodulation of cell homeostasis after contact with the tobaccocomposition from the second tobacco product as compared to the degree ofmodulation of cell homeostasis after contact with the tobaccocomposition from the first tobacco product identifies the second tobaccoproduct as one that has a reduced potential to contribute to atobacco-related disease. In some embodiments, the second tobacco producthas been modified to reduce a compound in the second tobacco. In someembodiments, the second tobacco product can be genetically modified toreduce the expression of at least one gene that regulates production ofthe compound. In some embodiments of the methods provided herein, thedegree of modulation of cell homeostasis can be determined byidentifying the degree of modulation of apoptosis. In some embodimentsof the methods provided herein, the degree of modulation of cellhomeostasis can be determined by identifying the degree of modulation ofcell proliferation. In some embodiments, the tobacco composition can besmoke or smoke condensate.

Also provided herein are methods of making a tobacco product that has areduced potential to contribute to a tobacco-related disease byproviding a first tobacco, obtaining a tobacco composition from thefirst tobacco, contacting a first population of cells with the tobaccocomposition from the first tobacco, identifying the degree of modulationof cell homeostasis in the first population of cells after contact withthe tobacco composition from the first tobacco, providing a secondtobacco, obtaining a tobacco composition from the second tobacco,contacting a second population of cells with the tobacco compositionfrom the second tobacco, identifying the degree of modulation of cellhomeostasis after contact with the tobacco composition from the secondtobacco product, where an identification of a reduction in the degree ofmodulation of cell homeostasis after contact with the tobaccocomposition from the second tobacco as compared to the degree ofmodulation of cell homeostasis after contact with the tobaccocomposition from the first tobacco identifies the second tobacco as onethat has a reduced potential to contribute to a tobacco-related disease,and incorporating the second tobacco, which has a reduced potential tocontribute to a tobacco-related disease, into a tobacco product. In someembodiments, the second tobacco has been modified to reduce a compoundin the second tobacco. In some embodiments, the second tobacco can begenetically modified to reduce the expression of at least one gene thatregulates production of the compound. In some embodiments of the methodsprovided herein, the degree of modulation of cell homeostasis can bedetermined by identifying the degree of modulation of apoptosis. In someembodiments of the methods provided herein, the degree of modulation ofcell homeostasis can be determined by identifying the degree ofmodulation of cell proliferation. In some embodiments, the tobaccocomposition can be smoke or smoke condensate.

The methods provided herein can be used to determine the effect of atobacco product or a compound from a tobacco product, on cellhomeostasis. Cells of an organism contacted with a tobacco composition,e.g., mammalian epithelial cells, can undergo apoptosis and canproliferate at particular levels under “normal” conditions, where“normal” as used in this context refers to conditions in which cells arenot contacted with tobacco or a tobacco composition and are nototherwise placed under atypical (e.g., stressful) environmentalconditions. Environmental conditions, for example, contacting the cellswith a tobacco composition, can modulate apoptosis of the contactedcells and also can modulate the proliferation of the contacted cells.Such modulation can result in processes that can directly lead tocellular events in tobacco-related disease (e.g., apoptosis can bedecreased, which can lead to neoplastic cell growth) or can indirectlylead to cellular events in tobacco-related disease (e.g., apoptosis canbe increased, which can trigger a cell growth response in an organism,which can lead to neoplastic cell growth). The methods provided hereincan be used to examine the affect of a tobacco product or a compoundfrom a tobacco product, on cell homeostasis by, for example, determiningthe affect of a tobacco or tobacco compound on apoptosis in a cell or acell population, or, for example, determining the affect of a tobacco ortobacco compound on cell proliferation of a cell or a cell population.In some embodiments, a first tobacco that causes a lesser degree ofmodulation of cell homeostasis relative to a second tobacco can becharacterized as a reduced risk tobacco. In some embodiments, a firsttobacco that causes a lesser degree of inhibition of apoptosis relativeto a second tobacco can be characterized as a reduced risk tobacco. Insome embodiments, a first tobacco that causes a lesser degree ofinhibition of cell proliferation relative to a second tobacco can becharacterized as a reduced risk tobacco. Any of a variety of knownmethods for determining modulation of cell homeostasis by, for example,modulating apoptosis, modulating cell proliferation, modulating geneexpression (e.g., mRNA or protein levels) as exemplified herein, can beused in the methods provided herein.

Also provided herein are methods for determining cell response to celldamage. Cells can be exposed to environmental input, such as a tobaccocomposition, that causes cell damage. The response of these cells to theenvironmental input-mediated damage can be indicative of the likelihoodof the environmental input leading to an environmental input-relateddisease. In one embodiment, cells can be contacted with a tobaccocomposition, and the response of the cells to the contact by the tobaccocomposition can indicate the likelihood of the tobacco compositionleading to a tobacco-related disease.

As provided herein, cells contacted by different environmental inputs,for example, different tobacco compositions, can respond differently tocell damage caused by the environmental input, where some cell responsesare more indicative of leading to a disease state compared to other cellresponses. Thus, contemplated herein, two or more tobacco compositionscan be compared and characterized according to the cell responses inreaction to damage induced by exposure to the tobacco compositions. Insuch methods, exposure conditions can be manipulated such that theamount of damage to the cells is equivalent for each different tobaccocomposition, resulting in a determination of different characteristiccell responses to the same amount of cell damage.

Accordingly, methods are provided herein for comparing two or moretobacco compositions by contacting a first tobacco composition with afirst population of cells, and contacting a second composition with asecond population of cells, where the two different contacting steps areperformed in such a manner that the first and second population of cellsundergo equivalent amount of cell damage, and then determining thedegree of modulation of cell homeostasis in the first and secondpopulations of cells, where the tobacco composition that ischaracterized by the lowest degree of cell modulation can be identifiedas a tobacco with reduced likelihood of causing a tobacco-relateddisease. In such methods, damage to the cells caused by the tobaccocompositions can be measured by, for example, measuring the degree ofdamage to the genetic material of the cells, in accordance with themethods provided herein or otherwise known in the art. Also in suchmethods, the degree of modulation of cell homeostasis can be determinedby the degree of modulation of apoptosis or cell proliferation relativeto cells not contacted by a tobacco composition or relative to cellscontacted by a tobacco composition from a tobacco, such as a reducedrisk tobacco with a known degree of modulation of cell homeostasis. Thefollowing section describes several methods for differentiating tobaccosand tobacco products that induce genetic damage from those that do not.

Analysis of Changes to Cell Homeostasis: Identifying a Tobacco thatInduces Genetic Damage

Provided herein are methods of identifying a tobacco that inducesgenetic damage by providing a tobacco, obtaining a tobacco compositionfrom the tobacco, contacting a cell with the tobacco composition, andidentifying the presence or absence of damage of cellular geneticmaterial in the cell after contact with the tobacco composition. In someembodiments, the methods provided herein can be used to identify atobacco that induces a double-strand DNA break. In some embodiments, thetobacco composition can be smoke or smoke condensate.

Also provided herein are methods of identifying a compound in tobaccothat induces damage of cellular genetic material by providing a firsttobacco, obtaining a tobacco composition from the first tobacco,contacting a first population of cells with the tobacco composition fromthe first tobacco, identifying the amount of damage of cellular geneticmaterial in the first population of cells after contact with the tobaccocomposition from the first tobacco, providing a second tobacco that hasbeen modified to reduce a compound in the second tobacco, obtaining atobacco composition from the second tobacco, contacting a secondpopulation of cells with the tobacco composition from the secondtobacco, and identifying the amount of damage of cellular geneticmaterial after contact with the tobacco composition from the secondtobacco, where an identification of a reduction in the amount of damageof cellular genetic material after contact with the tobacco compositionfrom the second tobacco identifies the compound as one that induces thedamage of cellular genetic material. In some embodiments, the methodsprovided herein can be used to identify a compound in tobacco thatinduces a double-strand DNA break. In some embodiments, the tobaccocomposition can be smoke or smoke condensate.

Also provided herein are methods of identifying a tobacco product thathas a reduced potential to contribute to a tobacco-related disease byproviding a first tobacco product, obtaining a tobacco composition fromthe first tobacco product, contacting a first population of cells withthe tobacco composition from the first tobacco product, identifying theamount of damage of cellular genetic material in the first population ofcells after contact with the tobacco composition from the first tobaccoproduct, providing a second tobacco product, obtaining a tobaccocomposition from the second tobacco product, contacting a secondpopulation of cells with the tobacco composition from the second tobaccoproduct, and identifying the amount of damage of cellular geneticmaterial after contact with the tobacco composition from the secondtobacco product, where an identification of a reduction in the amount ofdamage of cellular genetic material after contact with the tobaccocomposition from the second tobacco product as compared to the amount ofdamage of cellular genetic material after contact with the tobaccocomposition from the first tobacco product identifies the second tobaccoproduct as one that has a reduced potential to contribute to atobacco-related disease. In some embodiments, the second tobacco producthas been modified to reduce a compound in the second tobacco. In someembodiments, the second tobacco product can be genetically modified toreduce the expression of at least one gene that regulates production ofthe compound. In some embodiments of the methods provided herein, theamount of damage of cellular genetic material can be determined byidentifying the induction of double-strand DNA breaks. In someembodiments, the tobacco composition can be smoke or smoke condensate.

Also provided herein are methods of making a tobacco product that has areduced potential to contribute to a tobacco-related disease byproviding a first tobacco, obtaining a tobacco composition from thefirst tobacco, contacting a first population of cells with the tobaccocomposition from the first tobacco, identifying the amount of damage ofcellular genetic material in the first population of cells after contactwith the tobacco composition from the first tobacco, providing a secondtobacco, obtaining a tobacco composition from the second tobacco,contacting a second population of cells with the tobacco compositionfrom the second tobacco, identifying the amount of damage of cellulargenetic material after contact with the tobacco composition from thesecond tobacco product, where an identification of a reduction in theamount of damage of cellular genetic material after contact with thetobacco composition from the second tobacco as compared to the amount ofdamage of cellular genetic material after contact with the tobaccocomposition from the first tobacco identifies the second tobacco as onethat has a reduced potential to contribute to a tobacco-related disease,and incorporating the second tobacco, which has a reduced potential tocontribute to a tobacco-related disease, into a tobacco product. In someembodiments, the second tobacco has been modified to reduce a compoundin the second tobacco. In some embodiments, the second tobacco can begenetically modified to reduce the expression of at least one gene thatregulates production of the compound. In some embodiments of the methodsprovided herein, the amount of damage of cellular genetic material canbe determined by identifying the induction of double-strand DNA breaks.In some embodiments, the tobacco composition can be smoke or smokecondensate.

Also provided herein are methods, compositions and kits for evaluatingthe ability of a tobacco-derived substance to produce DSBs inchromosomal DNA. The presence of DSBs is detected using an appropriatemarker, which, in preferred embodiments provided herein, isphosphorylated histone H2AX (also referred to herein as “γH2AX”). Thepresence of DSBs also can be detected by detecting (i) activation of oneor more of the protein kinases that are responsible for H2AXphosphorylation (e.g., ATM, ATR and/or DNA-PK); (ii) appearance ofnuclear foci that are induced by histone H2AX phosphorylation; or (iii)activation of one or more protein components of nuclear foci induced byH2AX phosphorylation that are associated with DNA repair. The term“activation” in regard to proteins activated by DSBs refers to achemical modification such as phosphorylation, acetylation,ubiquitinylation or poly(ADP)ribosylation, and/or a change in proteinconformation, occurring in response to formation of DSBs. Activatedproteins can be detected, for example, immunocytochemically.

Some of the assays provided concern methods of detecting, quantifying,identifying and/or evaluating (e.g., for harmfulness) a tobacco-derivedsubstance in the course of research or in the environment via itspromotion of DSBs in the chromosomal DNA of a test cell. A correlationwith harmful potential is drawn based upon the known relationshipbetween DSBs and genetic mutations (including cancer-causing andteratogenic mutations) as well as cell damage and death.

Accordingly, one set of preferred embodiments provided herein aremethods of detecting a harmful tobacco-derived substance comprising thesteps of (a) exposing a test cell (or test cell population) to a tobaccotest composition; (b) measuring the degree of H2AX phosphorylation inthe test cell or cell population; and (c) comparing the degree of H2AXphosphorylation determined in the test cell or cell population to thedegree of H2AX phosphorylation in a control cell or control cellpopulation; wherein a higher degree of H2AX phosphorylation in the testcell compared to the control cell indicates the presence of a harmfultobacco derived substance in the tobacco test composition. The presenceof DSBs also can be detected by detecting (i) activation of one or moreof the protein kinases that are responsible for H2AX phosphorylation(e.g., ATM, ATR and/or DNA-PK); (ii) appearance of nuclear foci that areinduced by histone H2AX phosphorylation; or (iii) activation of one ormore protein components of nuclear foci induced by H2AX phosphorylationthat are associated with DNA repair.

Another set of non-limiting embodiments, provided herein include methodsfor identifying one or more harmful components of TS comprising thesteps of: (a) exposing a first test cell population to a first smokeproduct generated from a first tobacco composition; (b) exposing asecond test cell population to a second smoke product generated from asecond tobacco composition, wherein the first and second smoke productsare prepared using essentially equivalent protocols; (c) measuring thedegree of H2AX phosphorylation in the first and second test cellpopulations; and (d) comparing the degree of H2AX phosphorylation in thefirst and second test cell populations; (e) identifying the tobaccocomposition associated with a greater degree of H2AX phosphorylation insteps (a)-(d); and (f) comparing the components of the first and secondtobacco composition to identify one or more component present in thetobacco composition of step (e) but absent in the other tobaccocomposition. Methods for detecting activation of protein kinases such asATM, ATR and/or DNA-PK as well as formation of nuclear foci and proteincomponents of the nuclear foci can be performed according to the samesteps. According to such embodiments, the first tobacco composition candiffer from the second tobacco composition in its ingredients and/or inthe way it was processed. The information obtained by this method can beused to develop a tobacco product that lacks or has lower levels of theidentified harmful component(s), which can render the productlower-risk. Alternatively, the information can be used in anenvironmental context: for example, air purifiers can be modified toextract the harmful component from smoke-contaminated air.

Another set of non-limiting embodiments provided herein concern methodsfor identifying one or more harmful components of TS comprising thesteps of: (a) exposing a first test cell population to a first smokeproduct generated from a tobacco composition; (b) exposing a second testcell population to a second smoke product generated from the tobaccocomposition, wherein the first and second smoke products are prepareddifferently; (c) measuring the degree of H2AX phosphorylation in thefirst and second test cell populations; (d) comparing the degree of H2AXphosphorylation in the first and second test cell populations; and (e)identifying the method of smoke product preparation associated with agreater degree of H2AX phosphorylation in steps (a)-(d); wherein themethod of smoke product preparation identified in step (e) has greaterharmful potential. Methods for detecting activation of protein kinasessuch as ATM, ATR and/or DNA-PK as well as formation of nuclear foci andprotein components of the nuclear foci can be performed according to thesame steps. In such embodiments, the methods of smoke productpreparation can differ in the rate of combustion of the tobaccocomposition (including whether the tobacco composition is burned orheated), or can differ in the filtering of the smoke product (e.g.,unfiltered, filtered with a traditional filter, or filtered with afilter containing an antioxidant), or can differ by other known methodsof altering tobacco smoke products. The components of the differentsmoke products can be compared to identify one or more harmfulcomponents. As above, the identification of a harmful component canfacilitate the development of lower-risk tobacco products and/orenvironmental safeguards.

Also provided herein are methods for comparing the harmful potentials ofa first and a second tobacco composition comprising the steps of: (a)exposing a first test cell population to a first smoke product generatedfrom the first tobacco composition; (b) exposing a second test cellpopulation to a second smoke product generated from the second tobaccocomposition, wherein the first and second smoke products are preparedusing essentially equivalent protocols; (c) measuring the degree of H2AXphosphorylation in the first and second test cell populations; and (d)comparing the degree of H2AX phosphorylation in the first and secondtest cell populations; wherein the tobacco composition which generatedthe smoke product that produced a higher degree of H2AX phosphorylationhas greater harmful potential. Methods for detecting activation ofprotein kinases such as ATM, ATR and/or DNA-PK as well as formation ofnuclear foci and protein components of the nuclear foci can be performedaccording to the same steps.

Accordingly, the methods provided herein include one or more steps ofdetermining whether damage of cellular genetic material has occurred.Typically, such methods include assays for damage to the genomic DNA ofthe cell. Any of a variety of methods known in the art for assayingdamage of cellular genetic material, such as genomic DNA, can be used inthe methods provided herein. Exemplary known assays include assays fordouble-strand DNA breaks, assays for single-strand DNA breaks, andassays for modulated properties of DNA resultant from damage, such asassays for micronuclei and assays for chromosome exchange. Assays forDNA breaks are known in the art, as exemplified in U.S. Pat. Pub. No.20040132004 and U.S. Pat. No. 6,309,838, all of which are herebyexpressly incorporated by reference in their entireties.

In one example, the methods provided herein can include detection ofdouble-strand DNA breaks by detection of phosphorylation of histoneH2AX. Mammalian cells respond to agents that introduce DNAdouble-stranded breaks with the immediate and substantialphosphorylation of histone H2AX. While not wishing to be bound to thefollowing theory, which is only offered to explain one possiblemechanism, H2AX is thought to be involved in the recognition of regionsof chromatin containing a DNA double-stranded break. Formation of thephosphorylated H2AX protein, termed gamma-H2AX, can be detected as anindicator of DNA double-stranded breaks. Known antibodies orantigenically-reactive fragments thereof that specifically bind to aC-terminal phosphorylated serine in an H2AX histone protein can be usedfor the detection of gamma-H2AX, and, thus can be used to indicate thepresence of double stranded breaks in a cell. Thus, in the methodsprovided herein, the presence or absence of DNA damage can be detectedby detecting the presence or absence of phosphorylation of histone H2AX.For example, the presence or absence of phosphorylation of histone H2AXcan be identified with an antibody or fragment thereof, which binds tophosphorylated H2AX but not unphosphorylated H2AX. Antibodies andfragments thereof, and related methods for selectively detectinggamma-H2AX, are known in the art, as exemplified in U.S. Pat. Nos.6,362,317 and 6,884,873, all of which hereby expressly incorporated byreference in their entireties.

In some embodiments provided herein, the methods include assaying a cellfor double-strand DNA breaks (DSBs). DSBs are generated by a variety ofgenotoxic agents, and are among the most critical lesions that leadeither to apoptosis, mutations or the loss of significant sections ofchromosomal material. Detection of DSBs upon cell exposure to apotential carcinogen, therefore, provides the means to assess thepotential hazard of the exposure in terms of cancer induction. In oneembodiment, a sensitive assay of DSBs detection based on analysis ofhistone H2AX phosphorylation can be used. Histone H2AX, a variant of afamily of at least eight protein species of the nucleosome core histoneH2A, becomes phosphorylated in live cells upon induction of DNA doublestrand breaks (DSBs). The phosphorylation of H2AX on Ser 139 at sitesflanking the DSBs is carried out by ATM-, ATR-, and/or DNA-dependentprotein kinases (DNA-PKs). The phosphorylated form of H2AX is denotedγH2AX.

The availability of antibodies to γH2AX allow for immunocytochemicaldetection of DSBs. After induction of DSBs, the appearance of γH2AX inchromatin manifests in the form of discrete foci, each focus consideredto represent a single DSB. Checkpoint and DNA repair proteins such asRad50, Rad51 and Brcal co-localize with γH2AX. The intensity of γH2AXimmunofluorescence (IF) measured by cytometry was reported to stronglycorrelate with the dose of ionizing radiation and thus with the numberof the induced DSBs. However, because untreated cells, particularlycells replicating DNA, express γH2AX, to obtain a stoichiometricrelationship between DSBs and the intensity of γH2AX IF, it is necessaryto compensate for the extent of this “programmed” H2AX phosphorylation.Following compensation, the γH2AX IF measured by cytometry offers asensitive and convenient means to detect and measure DSBs in individualcells following radiation. In fact, γH2AX IF can be a surrogate for cellkilling in viability assays of radiated cells.

γH2AX antibody (“Ab”) in conjunction with multiparameter flow- and laserscanning cytometry can be used in assays of DSBs, to detect and measuretheir induction in individual, live cancer cells exposed to antitumordrugs in vitro. The intensity of γH2AX IF correlates well with the drugconcentration and duration of cell exposure to the drug, indicating arelationship between the incidence of DSBs induced by these drugs andγH2AX IF intensity. Multiparameter analysis of γH2AX IF and cellular DNAcontent made it possible to relate the abundance of DSBs (extent of DNAdamage) to the position of the cell in the cycle.

The ability of the tobacco-derived substance to promote the formation ofDSBs is measured using an appropriate DSB marker, which is preferablyγH2AX (phosphorylated histone H2AX), but which can be another associatedmolecule, such as, but not limited to, Rad50, Rad51 and Brcal, and otherproteins that are characteristic of nuclear foci formation. Formation ofDSBs also can be detected by detecting activate protein kinasesassociated with DSBs such as ATM, ATR or DNA-PK. The presence of suchmarkers can be determined using a marker-specific antibody (orderivative or fragment thereof), preferably an antibody (or fragment orderivative thereof) specific for γH2AX, or an antibody (or fragment orderivative thereof) specific for Rad50, Rad51 or Brcal, or ATM, ATR orDNA-PK. The presence of such markers can be determined using amarker-specific antibody (or derivative or fragment thereof), preferablyan antibody (or fragment or derivative thereof) specific for apolypeptide encoded by a gene provided in Tables 1 and 2. The genesprovided in Table 4 encode polypeptides that are involved in homologousrecombination processes in the cell, and these genes can be activated inresponse to cellular damage of genetic material. Accordingly, detectionof one or more products of the genes of Table 4 can be indicative ofcellular damage of genetic material, for example, double-strand DNAbreaks. The genes provided in Table 5 encode polypeptides that areinvolved in non-homologous nucleic acid end-joining processes in thecell, and these genes can be activated in response to cellular damage ofgenetic material. Accordingly, detection of one or more products of thegenes of Table 5 can be indicative of cellular damage of geneticmaterial, for example, double-strand DNA breaks. Provided herein is anexemplary use of antibody directed to γH2AX; analogous methods can beapplied using antibodies directed to Rad50, Rad51, Brcal, ATM, ATR orDNA-PK, or the products of the genes listed in Tables 1 and 2. Inpreferred non-limiting embodiments provided herein, antibody binding canbe detected by immunofluorescence-based techniques. Various antibodiesfor Rad50, Rad51, Brcal, ATM, ATR, DNA-PK, and the products of the geneslisted in Tables 1 and 2 are known in the art and can be readilyobtained for use in accordance with the methods provided herein; forexample, Anti-phospho-ATM (Ser1981), is available from Upstate USA asclone 10H11.E12. Such techniques can optionally be used in conjunctionwith automated cytometry, such as, for example, flow and/or laserscanning cytometry.

TABLE 4 Homologous recombination Top of Page RAD51 Homologous pairing15q15.1 NM_002875 RAD51L1 Rad51 homolog 14q24.1 NM_002877 (RAD51B)RAD51C Rad51 homolog 17q23.2 NM_002876 RAD51L3 Rad51 homolog 17q12NM_002878 (RAD51D) DMC1 Rad51 homolog, meiosis 22q13.1 NM_007068 XRCC2DNA break and crosslink 7q36.1 NM_005431 XRCC3 repair XRCC2, XRCC314q32.33 NM_005432 RAD52 Accessory factors for 12p13.33 NM_002879 RAD54Lrecombination 1p34.1 NM_003579 RAD54B RAD52, RAD54L, RAD54B 8q22.1NM_012415 BRCA1 Accessory factor for 17q21.31 NM_007295 transcriptionand recombination, E3 Ubiquitin ligase BRCA2 Cooperation with RAD51,13q13.1 NM_000059 essential function SHFM1 BRCA2 associated 7q21.3NM_006304 (DSS1) RAD50 ATPase in complex with 5q23.3 NM_005732 MRE11A,NBS1 MRE11A 3′ exonuclease 11q21 NM_005590 NBS1 Mutated in Nijmegen8q21.3 NM_002485 breakage syndrome MUS81 A structure-specific DNA11q13.1 NM_025128 EME1 nuclease MUS81, MMS4 17q21.33 NM_152463 (MMS4L)

TABLE 5 Non-homologous end-joining G22P1 (Ku70) 22q13.2 NM_001469 XRCC5(Ku80) 2q35 NM_021141 PRKDC 8q11.21 NM_006904 LIG4 13q33.3 NM_002312XRCC4 5q14.2 NM_003401 DCLRE1C (Artemis) 10p13 NM_022487

The term “immunofluorescence-based techniques” or“immunocytochemical-based techniques” encompasses various forms of suchassays, as are known in the art. For example, and not by way oflimitation, an immunofluorescence-based technique can use an unlabelledprimary antibody and a fluorescently labeled secondary antibody (asillustrated, for example, in Example 1); or can use a primary antibodythat carries a fluorescent tag to detect the phosphorylated H2AXmolecule directly; or the primary antibody can carry a biotin moleculewhile the secondary antibody can carry both an avidin molecule (whichbinds specifically to biotin) and a fluorescence molecule. In thebiotin/avidin approach, the binding of the secondary antibody is basedon binding of biotin by avidin rather than the binding of an antibody ofone species directed against a protein of another species. Othervariations of such techniques that would be known to the skilled artisanas “immunofluorescence-based techniques” or “immunocytochemical-basedtechniques” can be used according to the invention. Likewise, detectioncan be made using analogous methods that utilize a modality other thanfluorescence, such as chromogenic or colorimetric assays, radiologicassays, and so forth.

Techniques such as immunocytochemical-based techniques can be used inconjunction with methods for counting cells, sorting cells, or othermethod for further characterizing cells. Exemplary methods include, butare not limited to, flow cytometry, laser scanning cytometry,fluorescence image analysis, chromogenic product imaging, fluorescencemicroscopy or transmission microscopy.

The “degree of phosphorylation of H2AX” as used herein refers to therelative, rather than absolute, amount of γH2AX. This is because γH2AXis produced during normal progression of the cell cycle. As discussed inExample 1, allowance can be made for normally occurring phosphorylationof H2AX. For example, the data can preferably be subjected to twonormalization processes. First, allowance can be made for the normallyoccurring “programmed” phosphorylation of H2AX. Second, correction canbe made for the fact that histone content is exactly doubled over thecourse of a cell cycle, doubling the size of the target (histone). In aspecific non-limiting embodiment, a data value from cells with twice theDNA content (e.g., G2 and mitotic cells) with twice the histone targetcan be divided by 2 while a data value from cells in S phase having anintermediate in histone content can be divided by 1.5. In this manner,the amount of γH2AX detected beyond what occurs in an untreated controlcell or cell population is normalized to a unit of histone so that onecan refer to the “degree of histone H2AX phosphorylation” on a per unitof histone basis.

In another example, the methods provided herein can include detection ofDNA breaks and other forms of genomic damage by Comet assay. Comet assaycan be used to detect damaged DNA pulled from the nucleus of cellsexposed to an electric field. Comet assay is a fluorescent microscopicmethod to examine DNA damage and repair at individual cell level. Forexample, cells can be embedded in agarose on a microscope slide andlysed with detergent and high salt to form nucleoids containingsupercoiled loops of DNA linked to the nuclear matrix, andelectrophoresis at high pH can result in structures resembling comets,observed by fluorescence microscopy. The intensity of the comet tailrelative to the head reflects the number of DNA breaks. This assay canbe used for detecting various forms of DNA damage (e.g., single- anddouble-strand breaks, oxidative DNA base damage, andDNA-DNA/DNA-protein/DNA-Drug cross-linking) and DNA repair in manyeukaryotic cell types. Comet assay not only provides an estimate of howmuch damage is present in cells, but what form it takes. Although it isprimarily a method for measuring DNA breaks, modifications of themethods, for example, by introducing lesion-specific endonucleases,allows detection of, for example, pyrimidine dimers, oxidized bases, andalkylation damage. Thus, in the methods provided herein, the presence orabsence of DNA damage can be identified by, for example, the presence orabsence of comet tails when cells are analyzed using the Comet assay.Various methods for performing Comet assays are known in the art, asexemplified in Collins, (2004) Mol. Biotechnology 26:249-261, Tice, etal. (2000) Environ. Mol. Mutagen. 35:206-221 and Gichner et al. (2004)Mutation Res. 559:49-57, all of which are hereby expressly incorporatedby reference in their entireties.

In another example, the methods provided herein can include detection ofdouble-strand DNA breaks by TUNEL assay. TUNEL assay can be used tomeasure double-strand breaks by incorporation of labeled nucleotides atthe site of double-strand breaks using terminal transferase. The labelednucleotides can then be detected with antibodies. TUNEL assay isfrequently used to detect apoptosis-induced DNA fragmentation through aquantitative fluorescence assay. In one exemplary protocol, terminaldeoxynucleotidyl transferase (TdT) catalyzes the incorporation ofbromo-deoxyuridine (BrdU) residues into the fragmenting nuclear DNA atthe 3′-hydroxyl ends by nicked end labeling. A TRITC-conjugatedanti-BrdU antibody can then label the 3′-hydroxyl ends for detection.The TUNEL assay distinguishes two populations of cells: non-apoptoticcells (TUNEL-negative) and apoptotic cells (TUNEL-positive). Thus, inthe methods provided herein, the presence or absence of DNA damage canidentified by, for example, detecting the presence or absence of labelednucleotides at the site of double-strand breaks, incorporated by, forexample, terminal transferase. A variety of methods of performing TUNELassays is known in the art, as exemplified in Doolin et al., J. BurnCare Rehabil. 20: 374-376, 1999; Kalyuzhny (2002) Methods Mol. Biol.203:219-34; Lawry, Methods Mol. Med. (2004) 88:183-90; U.S. Pat. No.6,506,609 and U.S. Pat. Pub. No. 20030017462, all of which are herebyexpressly incorporated by reference in their entireties.

In another example, the methods provided herein can include detection ofdouble-strand DNA breaks by sister chromatid exchange assay. Sisterchromatid exchange assays detect late damage when genetic material isexchanged between sister chromatids. Sister chromatid exchange refers toa reciprocal interchange of the two chromatid arms within a singlechromosome. This exchange can be visualized during the metaphase portionof the cell cycle and can be mediated by the enzymatic incision,translocation and ligation of at least two DNA helices. Thus, in themethods provided herein, the presence or absence of DNA damage canidentified by, for example, detecting the presence or absence ofinterchange of chromatid arms within a single chromosome by, forexample, sister chromatid exchange assay. A variety of methods forperforming sister chromatid exchange assays are known in the art, asexemplified in 40 C.F.R. § 79.65, 40 C.F.R. § 798.5915, Renqing et al.,(2000) Toxicology Letters 115:23-32, Deen et al. and Cancer Res. (1986)46:1599-602, all of which are hereby expressly incorporated by referencein their entireties.

In another example, the methods provided herein can include detection ofdouble-strand DNA breaks by micronuclei assays. Micronuclei assays canbe used to detect late damage occurring after cells attempt to divide sothat non-centromeric DNA forms as micronuclei in daughter cells. Thetest is based on the observation that a secondary nucleus (micronucleus)is formed around a chromosomal fragment, outside the main nucleus of adividing cell. A micronucleus may also be produced due to a laggingwhole chromosome formed as a result of a chromosome loss at anaphase.Thus, in the methods provided herein, the presence or absence ofmicronuclei can be identified. Micronuclei can be detected bymicroscopic methods, flow cytometric methods and automated imagerecognition methods, as known in the art and exemplified in Offer etal., FASEB J. (2005) 19:485-7; Smolewski et al., Cytometry (2001)45:19-26; Driessens et al., Ann N Y Acad Sci. (2003) 1010:775-9; andU.S. Pat. Pub. No. 20050002552, all of which are hereby expresslyincorporated by reference in their entireties.

In another example, the methods provided herein can include detection ofchromosomal translocations. Chromosomal translocations can occur as aresult of DNA damage. Methods for detecting chromosomal translocationscan include fluorescence in situ hybridization methods (FISH), in whichprobe hybridization patterns in cells containing chromosomaltranslocation are altered relative to wild type. Thus, in the methodsprovided herein, the presence or absence of DNA damage can identifiedby, for example, detecting the presence or absence of chromosomaltranslocations by, for example, FISH. Methods for detecting chromosomaltranslocations are known in the art, as exemplified by U.S. Pat. Nos.5,997,869, 6,576,421, and 6,416,948, and U.S. Pat. Pub. Nos. 20040235039and 20020192692, all of which are hereby expressly incorporated byreference in their entireties.

The example below provides one non-limiting specific example of the DSBdetection methods and materials. Variations of the assay method used interms of materials, assay times, instrumentation and protocols would beapparent to the skilled artisan for detecting and/or quantifying DSBs,for example via γH2AX.

Example 1 Preparation of Cigarette Smoke Condensates

Smoke was generated from a commercially available nationally sold brandof American cigarettes (non-menthol, full-flavor type with averaged FTCmeasured values of 14.5 mg tar/1.04 mg nicotine) using an INBIFO-Condorsmoking machine under Federal Trade Commission (FTC) smoking parameters(2.0 second puff duration 35 milliliter puff every 60 seconds). Thecigarettes had been equilibrated at 23.9° C.±1.1° C. and 60%±2% relativehumidity for a minimum of 24 hours and a maximum of 14 days. CSC wascollected from the smoke via a series of three cold traps (−10° C., −40°C., and −70° C.) onto impingers filled with glass beads. The smokecondensate was dissolved in acetone, which was then removed by rotaryevaporation at 35° C. The resulting smoke condensate was weighed anddissolved in dimethylsulfoxide (DMSO) to make a stock solution at aconcentration of 20 mg/mL, which was stored at −20° C. prior to use.

NHBE Cell Culture and Smoke Condensate Treatment

NHBE cells were purchased from Cambrex Corporation, East Rutherford,N.J. The cells were cultured in complete Bronchial Epithelial CellGrowth Medium (BEGM), prepared by supplementing Bronchial EpithelialBasal Medium with retinoic acid, human epidermal growth factor,epinephrine, transferrin, triiodothyronine, insulin, hydrocortisone,bovine pituitary extract and gentamicin by addition of SingleQuots™(both medium and the supplements were purchased from CambrexCorporation, East Rutherford, N.J.). Dual-chambered slides (Nunc Lab-TekII, Fisher Scientific, Pittsburgh, Pa.) were seeded with 1 ml of 8×104cells/ml cell suspension per chamber. All incubations were at 37° C. ina humidified atmosphere of 5% CO2 in air. Cells were grown to 50%confluency, at which time they were treated with medium containing smokecondensate. Appropriate dilutions of the 20 mg/ml smoke condensate inDMSO stock solution were used to prepare culture medium containing 10,25, or 50 μg/mL smoke condensate. The final DMSO concentration was 0.5%.Cells were treated by carefully aspirating the culture medium from eachchamber and replacing it with 1 ml per chamber of smokecondensate-containing medium at 37° C. For control slides, the mediumwas replaced with 1 mL of either fresh medium (mock-treated control) ormedium containing 0.5% DMSO (vehicle control). Slides were immediatelyreturned to the incubator for up to 24 hours. At the end of thetreatment, medium from each chamber was carefully aspirated and 1 ml of1% fresh paraformaldehyde in 1× Dulbecco's PBS was added to each chamberand the cells fixed by gently rocking the slides at room temperature for15 minutes. Following aspiration of the fixative, the chamber slideswere disassembled and the slides submerged in 50 ml conical tubes filledwith 70% ethanol. The fixed slides were stored at 4° C. prior toanalysis.

A549 Cell Culture and Smoke Treatment

A549 cells were purchased from American Type Culture Collection (ATCC#CCL-185, Manassas, Va.). The cells were cultured in Ham's F12K mediumwith 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate(ATCC, Manassas, Va.) and supplemented with 10% fetal bovine serum(ATCC, Manassas, Va.). Dual-chambered slides (Nunc Lab-Tek II) wereseeded with 1 ml of 105 cells/ml cell suspension per chamber 48 hoursbefore exposure. All incubations were at 37° C. in a humidifiedatmosphere of 5% CO2 in air. Cells were grown to 70% confluency, atwhich time they were treated with smoke. The cell culture medium wasreplaced with 37° C. Dulbecco's PBS (D-PBS) containing calcium andmagnesium (Sigma, St. Louis, Mo.) for the smoke exposure. Slide chambercovers were removed and the slides were placed in a smoke exposurechamber (20.6 cm×6.7 cm×6.3 cm—L×W×H). Smoke was generated from IM16(Industry Monitor #16, Philip-Morris, Richmond Va.) cigarettes under FTCsmoking conditions using a KC 5 Port Smoker (KC Automation, Richmond,Va.). The smoke was diluted by drawing it through a 250 mL round-bottomflask prior to its reaching the exposure chamber. The time and distancethat the smoke traveled from the end of the cigarette to the exposurechamber was minimized by using the shortest lengths of tubing possiblebetween the parts of the apparatus. Cigarettes were smoked to within 3mm of the filter tip. Cells were exposed to smoke for up to 40 minutes.Mock-exposed (control) cells were treated under identical conditions asthe exposed cells except for the absence of a cigarette in the smokingport. They were mock-exposed for 10 minutes. Following treatment or mocktreatment, the D-PBS was aspirated and replaced with 1 ml per chamber offresh culture medium at 37° C. The slides were placed in the 37° C., 5%CO2 incubator and incubated for 15 minutes. Following incubation, themedium was aspirated and the cells fixed as described above for the NHBEexperiment.

Immunocytochemical Detection of Phosphorylated Histone H2AX andCaspase-3 Activation

Cells were treated with smoke (i.e., A549) or smoke condensate (i.e.,NHBE) and fixed as described above, then rinsed twice in PBS andimmersed in 0.2% Triton X-100 (Sigma) in a solution of 1% (w/v) bovineserum albumin (BSA; Sigma) in PBS for 30 min to suppress non-specificantibody binding. The cells were then incubated in 100 μl volume of 1%BSA containing 1:200 dilution of anti-phosphorylated histone H2AX(γ-H2AX) rabbit polyclonal Ab (Trevigen, Gaithersburg, Md.). Afterovernight incubation at 4° C., the slides were washed twice with PBS andthen incubated in 100 μl of 1:200 dilution of Alexa Fluor 488 goatanti-rabbit IgG (H+L) (Molecular Probes, Eugene, Oreg.) for 45 min atroom temperature in the dark. Parallel samples were incubated with 1:100diluted anti-cleaved (activated) caspase-3 rabbit polyclonal Ab (CellSignaling Technology, Beverly, Mass.) overnight at 4° C., washed twicewith PBS and incubated with 1:30 diluted FITC-conjugated F(ab′)2fragment of swine anti-rabbit immunoglobulin (DAKO, Carpinteria, Calif.)for 30 min in room temperature in the dark. The cells were thencounterstained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI,Molecular Probes, Eugene, Oreg.) in PBS for 5 min. Each experiment wasperformed with an IgG control in which cells were labeled only withsecondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (H+L) orFITC-conjugated F(ab′)2 fragment of goat anti-mouse immunoglobulins,without primary antibody incubation to estimate the extent ofnonspecific binding of the secondary antibody to the cells.

Measurement of Cell Fluorescence by Laser Scanning Cytometry

Cellular green (phosphorylated histone H2AX and cleaved caspase 3), andblue (DNA-bound DAPI) fluorescence emission was measured using a LaserScanning Cytometer (LSC; CompuCyte, Cambridge, Mass.), utilizingstandard filter settings; fluorescence was excited with 488-nm argon ionand violet diode lasers, respectively. The intensities of maximal pixeland integrated fluorescence were measured and recorded for each cell. Atleast 3,000 cells were measured per sample.

Statistical Analysis

To compare the changes in immunofluorescence intensity, the meanfluorescence intensity (integral values of individual cells) wascalculated for cells in each phase of the cycle by gating G1, S and G2/Mcells based on differences in DNA content. The means of the fluorescencevalue for G1, S and G2/M populations of cells in the IgG control groupswere then subtracted from the respective means of the smoke condensateor smoke-treated cells. All experiments were run under identicalinstrument settings. Data is presented as mean γH2AX fluorescence ofeach cell cycle compartment or where not indicated, of the entirepopulation (G1, S and G2M). Each experiment was run in duplicate ortriplicate. All experiments were repeated at least three times.

Filter Comparison

Tests for phosphorylated histone H2AX also were applied to tests ofseveral filters attached to different tobaccos. Filters and tobacco wereobtained from: (1) the industry standard reference tobacco IM16 (PhilipMorris® USA); (2) reduced risk cigarette Omni® (Vector Tobacco Ltd.);and (3) reduced risk cigarette Quest 3® (Vector Tobacco Ltd.). A549cells were exposed to mock treatment (control) and cigarette smokesubstantially as provided in the above smoke treatment description.

Each of IM16, Omni® and Quest 3® were tested, and the γH2AX (smoke-mock)time course for each is presented in FIG. 44A. FIG. 44A demonstratesthat each of IM16, Omni® and Quest 3® have clearly different influenceson γH2AX levels, where the γH2AX levels parallel the expected level ofrisk attributed to the tobacco product (IM16 is highest expected riskand has the highest γH2AX levels, while Quest 3® is lowest expected riskand has the lowest γH2AX levels).

Next, the influence of IM16, Omni® and Quest 3® filters were compared byconfiguring a cigarette with IM16 tobacco, and each of the IM16, Omni®and Quest 3® filters. FIG. 44B demonstrates that the cigaretteconfigured with the IM16 filter resulted in the highest γH2AX levels,while the cigarette configured with the Quest 3® filter resulted in thelowest γH2AX levels. Thus, when the same tobacco (IM16) is used, theγH2AX levels reflect the influence of the filter on the number of doublestranded DNA breaks caused by tobacco smoke. In the instant example,when IM16 tobacco is used, the γH2AX levels parallel the expected levelof risk attributed to the tobacco filter (IM16 is highest risk filterand has the highest γH2AX levels, while Quest 3® is lowest risk filterand has the lowest γH2AX levels).

Next, the filters were tested using Omni® and Quest 3® tobaccos. FIG.44C demonstrates that when Omni® tobacco is used, a cigarette containingthe IM16 filter results in comparable γH2AX levels as compared to acigarette containing the Omni® filter. Thus, FIG. 44C demonstrates thatthe risk-reducing properties of the tobacco and the risk-reducingproperties of the filter can be interrelated such that the risk-reducingproperties of a particular filter can vary depending on the type oftobacco used. FIG. 44D demonstrates that when Quest 3® tobacco is used,a cigarette containing the IM16 filter results in higher γH2AX levels ascompared to a cigarette containing the Quest 3® filter.

Exposure of A549 cells to TS induces H2AX phosphorylation, which can bedetected immunocytochemically (FIG. 12). Though the intensity of greenγH2AX IF varies from cell to cell, its distribution is nuclear andpunctuate. Mock-treated cells have minimal, but still detectable levelsof γH2AX IF.

FIG. 13 illustrates the raw data in the form of scattergrams of the A549cells untreated (0 time) and exposed to TS for 30 min. A scattergramrepresenting cells immunostained with an irrelevant isotype control IgGis also included in the figure. The intensity of fluorescence of themock-exposed cells is distinctly higher than that of the isotypecontrol. This is a reflection of the “programmed” phosphorylation ofH2AX, known to occur during normal progression through the cell cycle.Exposure of A549 cells to smoke, in this instance, markedly increasedcellular γH2AX IF. The increase, however, was proportional for the cellsin each phase of the cell cycle.

As noted above, the mean “programmed” H2AX IF was subtracted from themean γH2AX IF of the cells exposed to either smoke or smoke condensate,separately for cells in each phase of the cell cycle, for eachdata-point shown in the FIGS. 14 and 15. In addition, since the amountof histone doubles as cells proceed from G₁ to G₂ phase, γH2AX IF wasnormalized to DNA/histone content by dividing the mean γH2AX IF of the Sand G₂M phase cells by 1.5 and 2, respectively. The normalized data,therefore, does not represent the total amount of phosphorylated H2AXper cells but rather the degree of H2AX phosphorylation, independent ofthe increase in total H2AX IF that occurs during progression through S.

During the initial 10 min exposure of A549 cells to smoke, no change inγH2AX IF was apparent (FIG. 14). However, between 10 and 20 min exposureto smoke, γH2AX IF increased by 71%, 67.5% and 45.7% for G₁, S and G₂Mphase cells, respectively. An additional 10 min of exposure to smoke (30min in total) resulted in an additional increase in γH2AX IF compared tomock-exposed cells: 151.2%, 132.2% and 109.3% for G₁, S or G₂M phasecells.

The plots shown in FIG. 15 display the increase in the level of H2AXphosphorylation as a function of length of exposure of NHBE cells to 10,25 or 50 μg/ml concentrations of smoke condensate. At eachconcentration, the maximal rate of increase in H2AX IF was seen duringthe initial 4 h of cell treatment. However, whereas at 10 and 25 μg/mlof smoke condensate the peak of H2AX phosphorylation occurred at 4 h,followed by a plateau up to 24 h, at a smoke condensate concentration of50 μg/ml, H2AX phosphorylation increased during the entire 24 h timecourse of the experiment. No cell cycle phase specificity was apparentin H2AX phosphorylation when cells were exposed to 10 μg/ml smokecondensate (FIG. 16). The same was true for these cells exposed to 25 or50 μg/ml.

Activation of caspase-3 was measured in samples parallel to those thatwere subjected to analysis of H2AX phosphorylation, by detecting thepresence of activated caspase-3 immunocytochemically. Exposure of A549cells to smoke for up to 40 min followed by their fixation at 15 minuteshad no effect on caspase-3 activation: less than 0.5% of the cellsdemonstrated the presence of activated caspase-3 in either mock-exposedor smoke treated cultures (Table 6). Caspase-3 activation could beshown, however, if A549 cells exposed to smoke for 20 min were allowedto grow in culture for an extended period of time (24 h) at which pointvirtually half the cells were positive for activated caspase-3 (Table6).

TABLE 6 Effect of Smoke on Caspase-3 Activation Exposure to smoke Timein culture % Caspase-3 positive (min) following exposure (h) cells (%)*0 0.25 0.1 10 0.25 0.4 20 0.25 0.1 30 0.25 0.4 40 0.25 0.1 0 24 0.2 2024 49.9 *Caspase-3 positive cells were detected immunocytochemically, asdescribed elsewhere.

The present results demonstrate that exposure of A549 cells to TS orNHBE cells to TSC induces phosphorylation of H2AX. The extent of H2AXphosphorylation is concentration-dependent. It also correlated with theduration of exposure. In the case of NHBE cells, while at lower smokecondensate concentrations (10 and 25 μg/ml), a plateau is achieved after4 h, at 50 μg/ml concentration, progressive phosphorylation continuesfor up to 24 h. H2AX phosphorylation in the A549 cells exposed to smokealso progresses with time of exposure, although it appears to plateauafter 30 min. Phosphorylation of H2AX is a specific marker of inductionof DSBs; the present data indicate that TS and TSC both induce suchbreaks in A549 cells and NHBE cells in a dose and time dependent manner.

It should be noted that H2AX is intensely phosphorylated in response toDNA fragmentation that occurs upon induction of apoptosis. Caspaseactivation, however, is required to trigger apoptosis-related DNAfragmentation. In fact, inhibition of caspase-3 activity (e.g. byz-VAD-FMK) can prevent the apoptosis-associated H2AX phosphorylation. Inthe present study, no caspase-3 activation was detected in the cellsexposed for up to 40 min to smoke (Table 6). Thus, apoptosis-associatedphosphorylation of H2AX did not contribute to the γH2AX IF measured inA549 cells exposed to smoke for up to 40 min, when the cells werecollected within 15 min of exposure.

The present assay provides quantitative results. Specifically, thenumber of H2AX phosphorylation foci is considered to correspond to thenumber of DSBs. Assuming that the individual foci have comparableintensity of IF, the integrated value H2AX IF, as presently measured,would be expected to correspond to the number of foci, hence, to thenumber of DSBs. Furthermore, the mean γH2AX IF of the mock-exposed cellswas subtracted from each mean of cells exposed to smoke or smokecondensate, to ensure that the measurement was not affected by the levelof “programmed” H2AX phosphorylation in these cells (see FIG. 13).Though not applicable in the present instance in which the time betweenexposure to smoke or smoke condensate and harvesting of the cells wasrelatively short (55 min or less), a phosphatase inhibitor such ascalyculin A or okadaic acid can be included in the culture to preventpossible dephosphorylation of H2AX molecules. The data presented in theplots, therefore, represent the smoke-induced differential γH2AX IF.Furthermore, since the H2AX content increases as cells traverse throughS phase, the mean values γH2AX IF for S and G₂/M cells were compensatedfor the H2AX increase. The intensity of γH2AX IF so compensated, thus,reflects the degree of H2AX phosphorylation in the cell, i.e. isunrelated to H2AX content.

There is little evidence that CS and specific smoke constituents cancause single strand breaks (SSBs) in the normal human genome, but noevidence for the induction of DSBs. DSBs are among the most deleterioustypes of DNA damage in mammalian cells. A cell that incurs DSBs is atmajor risk for developing genomic instability, which can result in anarray of specific defects such as chromosome fragmentation,translocation, rearrangement and loss. More importantly, each of thesechromosomal abnormalities can play a pivotal role in the etiology orprogression of a wide range of human cancers. Consequently, in order toensure the faithful repair of DSBs and maintain genomic integrity, thecell has evolved sensitive DNA damage-activated checkpoint controlpathways that are coupled to an interconnected web of efficient repairmechanisms, the most prominent of which are homologous recombination andnon-homologous end joining. Individuals who either have debilitatingalterations or deletions of the genes involved in detecting andrepairing DSBs tend to manifest the dual syndromes of chromosomeinstability and higher incidence of various cancers. Clearly, therefore,the induction of DSBs by an exogenous agent like TS can be a potentiallyhazardous genetic event in the long-term smoker. In particular, ifoverall repair efficiencies of DSBs are not as efficient as for othertypes of DNA damage, e.g., single strand breaks (SSBs), and/or if anindividual smoker has specific polymorphisms in the relevant genes thatreduce their effectiveness, then cells chronically exposed to TS canmanifest the genetically dangerous combination of increased levels ofDSBs and compromised repair capacities. Furthermore, in addition to DSBlevel and repair capacity, the genomic positioning of DSBs can beanother factor that determines how successfully a cell responds to thistype of damage. For example, the probability that a DSB break isinaccurately rejoined is relatively low when DSBs are spatiallyseparated but increases considerably when multiple breaks coincide.

The successful repair of DSBs appears also to depend on cell cycleposition. The data, however, show no obvious cell cycle specificity interms of accumulation of DSBs. Thus, if proliferating cells exposed toTS experience similar levels of DSBs during each phase of the cell cyclebut dissimilar repair rates, they can be particularly susceptible toaccumulating deleterious DNA defects during that specific phase. It isrelevant to point out that although the rates of DSB induction andrepair in noncycling cells, which are one of the initial primary targetcells in lungs exposed to CS, can be different than in cycling cells,the lungs of persistent smokers undergo a significant increase in thenumber of proliferating cells due to smoke-induced damage. Moreover,cells actively dividing at the time of carcinogen exposure are atparticular risk for transformation-related events.

The methods of identifying a tobacco, identifying a compound in tobacco,identifying a tobacco product, and making a tobacco product providedherein, can additionally be used to compare two or more tobaccos so asto identify a toxic compound, evaluate the potential risk posed by thetobacco products, or to develop reduced risk tobacco products. In someembodiments, the two or more tobaccos are compared for their ability toinduce damage to the genetic material of cells. In some embodiments, atleast one tobacco is a reduced risk tobacco or identified as a reducedrisk tobacco. In some embodiments, at least one tobacco is a modifiedtobacco, such as a chemically modified tobacco or a genetically modifiedtobacco.

Example 2 below provides one non-limiting specific example of methodsfor comparing tobaccos in accordance with the methods provided herein.Variations of the assay method used in terms of assay methodologies(e.g., assay for apoptosis or for cell proliferation) would be apparentto the skilled artisan for comparing tobaccos.

Example 2

A549 cells were exposed to whole smoke from IM16 cigarettes for variouslengths of time, washed and allowed to grow for an additional hourbefore being harvested for analysis. DNA damage was identified as anincrease in phosphorylation of histone H2AX denoted as γH2AX.

In order to compare DNA damage as a function of the cell's position inthe cell cycle, γH2AX values were normalized to DNA content sincehistone content doubles as cells proceed from G1 to G2 phase. Thus, inorder to determine any change in histone H2AX phosphorylationindependent of changes in histone/DNA content or DNA ploidy, the valuesfor S and G2M phase populations, gated according to DNA content, weremultiplied by 0.75 and 0.5, respectively. In instances where“normalized” values of γH2AX are presented, these values were obtainedby subtracting the mean values of each cell cycle population (or thetotal population) from the mean of the mock-treated population whoseγH2AX values represent “scheduled” γH2AX expression. In all instances,the values presented represent the mean γH2AX fluorescence of thepopulation; typically 3-5×103 cells were analyzed for each condition.

As illustrated in FIG. 17, there was little or no change in γH2AX whenexposure of A549 cells to whole smoke was limited to 5 min. However, asthe time of exposure exceeded 5 min there was a more or less linearincrease in γH2AX. Initially, S phase cells appeared most sensitive toDNA damage expressing approximately 37% higher levels of γH2AX than G1phase cells following 10 min of exposure to smoke. When the length ofexposure was increased to 20 min, G1 phase cells invariably expressed10-20% higher levels of γH2AX-associated fluorescence.

In another set of experiments it was determined that the extent of DNAdamage varied with the length of time of recovery following exposure towhole smoke. Previous studies have shown that following exposure towhole smoke for times in excess of 20 min leads to a significantincrease in apoptotic cells in the population depending upon when theassay is performed. Apoptotic cells contained significantly increasedlevels γH2AX compared to what one sees when assessing the primary breaksdue to DNA damaging agents. Based on the absence of activation ofcaspase 3, there is little or no induction of apoptosis in A549 cellswithin the first 3 h following 20 min exposure to whole smoke from IM16.

Within 15 min of exposure to whole smoke, A549 cells already displayed adramatic increase in γH2AX relative to mock exposed cells (FIG. 18A).Increasing the recovery time following exposure led to continuedincrease in DNA damage. As noted above, G₁ cells appear to be the mostsensitive to smoke especially when the cells are harvested 30 min orlonger after exposure to whole smoke. The relative increase in γH2AXfollowing 60 min of recovery is illustrated in FIG. 18B where it can beobserved that virtually all smoke-exposed cells (right) express levelsof γH2AX in excess of the expression observed in the mock-treated cells(left).

The response of NHBE cells to whole smoke from IM16 cigarettes was moreor less identical to that observed for A549 cells (FIG. 18C). The onedifference between the two cell lines was that S phase cells in NHBEcultures always expressed higher “scheduled” amounts of γH2AX.Nevertheless, as with A549 cells, G₁ cells are the most sensitive tosmoke-induced DNA damage in these cultures. FIG. 18D demonstrates boththe increased basal level of γH2AX in mock-treated cultures (left) andthe extensive increase in γH2AX expression observed 60 min following a20 min exposure of cells to whole smoke (right).

In the next series of experiments, the DNA damage caused by whole smokefrom different sources was compared. Using an exposure time of 20 min,damage due to whole smoke from two other cigarettes could be compared tothat caused by IM16 following various recovery times. The curves ofγH2AX following exposure of A549 cells to IM16 (FIG. 19, top right) werecomparable to that displayed in FIG. 18A. Exposure of the same cells towhole smoke from Quest 3® on the other hand resulted in an initialincrease in γH2AX at 30 min that returned to near background levels whenassayed after longer recovery times (FIG. 19, bottom left). Whole smokefrom Omni® cigarettes caused damage intermediate between that of Quest3® and IM16 (FIG. 19, bottom right). The DNA damage caused by Omni®increased until 60 min after which it more or less plateaued. Smoke fromQuest 3® cigarettes affects S phase cells to a greater extent than anyother phase while G1 cells are invariably most sensitive to smoke fromIM16 and Omni®. Importantly, these data demonstrate that tobaccoproducts containing modified tobacco (i.e., Omni® and Quest 3®) inducedless DNA damage than a reference tobacco product (i.e., IM16).Accordingly, the modified tobacco products Omni®, and Quest 3® have areduced potential to contribute to a tobacco related disease (i.e.,Omni®, and Quest 3® are reduced risk tobacco products) according to thedouble strand break assay.

In the next series of experiments, it was determined that DNA damagecaused by whole smoke can be mitigated by the presence of NAC. Using astandardized set of conditions (20 min of exposure followed by a 1 hrecovery), DNA damage caused by whole smoke from IM16 cigarettes wasassayed in both A549 and NHBE cells. NAC at a concentration of 25 mM waseither absent or present during exposure and absent or present duringthe 1 h recovery time. In this instance, the background or “scheduled”γH2AX expression observed in Mock-treated cells was subtracted from eachmeasurement. The remaining fluorescence should be indicative of thelevel of DNA DSBs under each set of conditions.

In A549 cells (FIG. 20, top), IM16 caused a dramatic increase in H2AXphosphorylation in the absence of NAC (PBS, PBS). Applying NAC to themedia following exposure to smoke did nothing to mitigate the DNA damagecaused by whole smoke. However, if NAC was present during exposure tosmoke, DNA damage was suppressed by greater than 80% for the entirepopulation; the suppression was greatest for G1 cells (91%),intermediate for G2M (88%) and least for S (82%) phase cells. Thepresence of NAC both during exposure to smoke and during the 1 hrecovery period provided slightly more protection increasing suppressionof γH2AX to 90% for the entire population.

As with A549 cells, when NHBE cells were exposed to whole smoke fromIM16 cigarettes, the cells in G1 phase were the most sensitive. However,since the S phase cells express somewhat higher levels of “scheduled”γH2AX and are not as sensitive as G1 cells to smoke (FIG. 18C), thevalue for S phase cell DNA damage was considerably less than for cellsin G1 or G2M phase (FIG. 20, bottom). Addition of NAC only duringrecovery had little effect on the level of DNA damage induced by wholesmoke. NAC present during exposure diminished the damage observed in G1cells by nearly 69%; the decrease was about 65% for G2M cells but Sphase cells were afforded no protection. NAC present both duringexposure and recovery provided a small degree of additional protection.

Next, the effect of NAC on DNA damage caused by whole smoke from varioussources was evaluated. A549 cells were exposed to smoke from IM16, Omni®and Quest 3® cigarettes in the presence and absence of NAC duringexposure. As illustrated in FIG. 21, NAC dramatically reduced theeffects of smoke from IM16 cells. Omni® produced less damage than IM16but NAC reduced the damage to near background levels. Quest 3® smokecaused the least amount of damage which could also be reduced tobackground levels by the presence of 25 mM NAC during exposure. In allinstances, the level of damage following exposure to smoke in thepresence of NAC was approximately the same, just slightly more than thebackground or scheduled level of γH2AX expression. As above, the datafrom this assay demonstrates that tobacco products containing modifiedtobacco (i.e., Omni® and Quest 3®) induced less DNA damage than areference tobacco product (i.e., IM16). Again, the double strand breakassay has shown that the modified tobacco products Omni®, and Quest 3®have a reduced potential to contribute to a tobacco related disease(i.e., Omni®, and Quest 3® are reduced risk tobacco products).

In more experiments, the cell cycle specific inhibition of wholesmoke-induced DNA damage by NAC was analyzed. A549 cells were exposed towhole smoke in the presence and absence of various concentrations ofNAC. Exposure was always for 20 min and recovery was 1 h. In eachinstance, the background or “scheduled” expression of γH2AX wassubtracted from the value obtained for each population in each cellcycle phase. Since G1 phase cells were the most sensitive and had thehighest value, all other measurements were normalized to that of G1phase cells exposed to IM16 smoke in the absence of NAC (plotted as 0.1mM NAC on the log plot).

As can be seen in FIG. 22, damage by whole smoke from IM16 to S phaseA549 cells was unaffected by the presence of NAC up to a concentrationof 5 mM. In contrast, damage caused to both G1 and G2M cells began todecrease when as little as 1 mM NAC was present during exposure. Thedamage caused to S phase cells decreased sharply as the NACconcentration was increased to 10 mM and, by 25 mM, there was littledifference in residual γH2AX expression between cells in any phase ofthe cycle.

The concentration of NAC that reduced DNA damage by 50% for each cellcycle phase can be determined from the graph in FIG. 22. For G1, S andG2M phase cells the values were approximately 4.5, 2.6 and 7.5 mM NAC.

In more experiments, it was determined that the vapor phase of smokeinduces damage that is abrogated by the presence of NAC. FIG. 23 (top)illustrates the ability of the vapor phase of smoke from various tobaccosources to cause DNA damage to A549 cells in comparison to whole smokefrom IM16 cigarettes. Thus, the vapor phase from IM16 cigarettes usingstandard conditions of exposure and recovery caused only about 26% ofthe DNA damage (γH2AX) as whole smoke from the same source. In the samecomparison, the vapor phase from Quest 1® and Quest 3® caused only 8.1%and 5.6% of the damage caused by whole smoke from IM16. As a directcomparison, the vapor phase of smoke from Quest 1® and Quest® caused68.8% and 78.5%, respectively, less damage than the vapor phase of smokefrom IM16.

The presence of 25 mM NAC during exposure of A549 cells to whole smokeform IM16 cigarettes reduced γH2AX by nearly 90% (89.1%) compared tocells exposed to whole smoke in the absence of NAC. NAC present duringcell exposure to the vapor phase of smoke from IM16, Quest 1® and Quest3®, reduced γH2AX by 93.2%, 98.9% and 100%, respectively compared to thedamage caused by the vapor phase of smoke in the absence of NAC.

The same experiment performed on NHBE cells resulted in more or lesscomparable results (FIG. 23, bottom). Whole smoke from IM16 cellsproduced less damage in NHBE cells under standard conditions compared toA549 cells (note the greater background observed in NHBE cells). Thevapor phase from IM16 CS caused only about 30% (29.7%) of the damagecaused by whole smoke whereas the vapor phase of smoke from Quest 1®caused 97% less damage than whole smoke from IM16 cigarettes. The vaporphase of smoke from Quest 3® produced no increase in γH2AX overbackground in NHBE cells.

The presence of NAC during exposure of NHBE cells to whole smoke fromIM16 cigarettes reduced γH2AX by about 78% (77.9%). The presence of NACduring exposure of cells the vapor phase of IM16, Quest 1® or Quest 3®abolished virtually all DNA damage relative to mock-treated cells; i.e.,γH2AX was reduced to background levels or below.

The cell cycle phase specific results are comparable to that for thewhole populations (FIG. 24). The vapor phase of smoke from IM16 causedcomparable amounts of damage in each cell cycle phase in A549 cellsthough the reduction of damage in G1 phase by NAC was somewhat higherthan it was for S and G2M phase; 98.5% versus 89.0% and 92.2%,respectively. The vapor phase from both Quest 1® and Quest 3® causedmore damage to S phase cells though in each instance, the presence ofNAC reduced damage to background levels for each cell cycle phase.

NHBE cells as noted earlier have higher γH2AX levels in S phase ofmock-treated cells as can be seen in FIG. 24. The largest increase indamage caused by the vapor phase of smoke from IM16 occurred in G1 phasecells (54.4% and 66.9% greater than for cells in S or G2M,respectively). The presence of NAC reduced the damage caused by thevapor phase of smoke from IM16 to background levels or below. The vaporphase of smoke from Quest 1® and Quest 3® cigarettes had only a smalleffect on DNA damage in cells in G1 or S but not G2M phase. All damagecaused by the vapor phase of smoke from Quest® cigarettes in NHBE cellswas inhibited in the presence of NAC. Importantly, this data providemore evidence that the tobacco products containing modified tobacco(i.e., Quest 1® and Quest 3®) induced significantly less DNA damage(i.e., double strand DNA breaks) than that of a reference tobaccoproduct (i.e., IM16). Accordingly, the modified tobacco products Quest1®, and Quest 3® have a reduced potential to contribute to a tobaccorelated disease (i.e., Quest 1® and Quest 3® are reduced risk tobaccoproducts, according to the double strand DNA break assay.

FIGS. 30, 32 and 33 show additional comparisons of reactions of A549cells to smoke from various cigarettes, where the affect can vary fordifferent cigarettes, and can vary according to the cell cycle of thecells, and can vary according to the presence of antioxidant.

Further performed was a test of double-strand DNA breaks in the cells ofa human subject exposed to tobacco smoke. The level of γH2AX expressionin the buccal mucosa of a smoker was compared to the level of γH2AXexpression in the buccal mucosa of a nonsmoker. A cheek swab wascollected from a subject (smoker) within 5 min completion of smoking aMarlboro Light® cigarette, and a second check swab was collected from asubject that did not smoke a cigarette (non-smoker). Levels of γH2AXwere then measured for both cell samples. As seen in FIG. 31 the X axisdepicts γH2AX associated fluorescence (γH2AX), and the Y axis depictsthe number of cells having the corresponding γH2AX fluorescence level.There were 358 cells with a very low value of γH2AX in the non-smokersample, whereas the smoker sample had cells with γH2AX values spreadover a wide range. Each histogram represents 3×103 cells. The buccalcells from the smoker showed a low number of cells having little or noγH2AX fluorescence signal, and showed a large number of cells withhigher γH2AX fluorescence levels. In contrast, almost all cells of thenon-smoker had little or no γH2AX fluorescence. Thus, human buccal cellsexposed to tobacco smoke have an increased level of double strand DNAbreaks relative to human buccal cells not exposed to tobacco smoke.These results parallel the in vitro results observed for A549 cells andfor NHBE cells. Thus, the in vitro approaches described herein arepredictive of in vivo responses.

Accordingly, the methods that were applied to A549 cells and NHBE cellsfor comparing different tobacco products, analyzing cells at differentstages in cell cycle, and determining protection provided by thepresence of an antioxidant, will be performed on human samples of buccalcells and it is expected, as shown in the in vitro experiments, thatmodified tobaccos, in particular genetically modified tobaccos that havea reduced amount of one or more compounds that contribute to a tobaccorelated disease (e.g., genetically modified tobacco having a reducednicotine, TSNA, and/or sterol content) will induce fewer or a reducedamount of double strand DNA breaks in humans that are contacted withsmoke from said modified tobaccos than will be observed in humans thatare contacted with smoke from conventional tobacco products, referencetobacco products, or non-transgenic (wild-type tobacco of the samevariety as the parental strain prior to genetic modification). Thefollowing section describes several methods to evaluate the ability of atobacco or a tobacco product to modulate apoptosis in greater detail.

Analysis of Changes in Cell Homeostasis: Changes in the Fidelity of theDNA, Double Strand Breaks

By one approach, for example, CS is generated using a smoking machinefrom a first tobacco modified product, e.g., a product containingtobacco that has been genetically modified to have a reduced amount of acompound. A first population of NHBE cells is contacted with said CSobtained from the modified tobacco product, and the cells contacted withCS are assayed for double-strand DNA breaks. A second population of NHBEcells is then contacted with CS generated from an unmodified tobaccoproduct, wherein the unmodified tobacco product retains the componentthat was removed or inhibited in the modified tobacco product. Anunmodified tobacco product can be, for example a product containing theparental variety of tobacco, where the parental variety of tobacco isthe unmodified tobacco variety used to generate the modified tobaccovariety. The second population of cells contacted with CS is thenassayed for double-strand DNA breaks. A comparison of the data obtainedfrom the analysis of the first and second tobacco products will revealthat the difference in double-strand DNA breaks caused by the modifiedtobacco product relative to the unmodified tobacco product. By thisapproach, one can effectively identify the contribution of individualcomponents of a tobacco product to double-strand DNA breaks, or otherassay conditions provided herein. These methods can thereby be used toidentify the contribution of individual components of a tobacco productto a tobacco-related disease. This approach can be used to developtobacco products that are less likely to contribute to a tobacco-relateddisease and reduced risk tobacco products identified by these methodsare embodiments provided herein. Further, tobacco products prepared bythese approaches can be prepared according to good manufacturingprocesses (GMP) (e.g., suitable for or accepted by a governmentalregulatory body, such as the Federal Drug Administration (FDA), andcontainers that house said tobacco products can comprise a label orother indicia, with or without structure-function indicia, whichreflects approval of said tobacco product from said regulatory body.

Thus, the methods provided herein can be used to characterize a firstand a second tobacco product by providing the first and second tobaccoproducts, obtaining a first and second tobacco composition from thefirst and second tobacco products, respectively, contacting a first cellwith the first tobacco composition and contacting the second cell withthe second tobacco composition, and identifying one or more attributesof the contacted cells. Different tobacco products can contain differentlevels of carcinogens that can induce various types of cell damageincluding mutations, chromosomal aberrations, aberrant sister chromatidexchanges and micronuclei. Comparison of attributes of cells contactedwith different tobacco compositions can be performed in the methodsprovided herein, and such attributes include, but are not limited to,differential levels of mRNA, differential levels of protein, inductionof damage of cellular genetic material or modulation of cellhomeostasis. Accordingly, the methods provided herein can be used tocompare two or more tobacco products by assay methods including assayfor differential levels of mRNA, differential levels of protein,induction of damage of cellular genetic material or modulation of cellhomeostasis. Exemplary assay methods include microarray assays, ELISAassays, Western blot assays, assays of a double-strand DNA break,inhibition of apoptosis, or inhibition of cell proliferation.

In some embodiments, the first and second smoke products are preparedusing essentially equivalent protocols. The phrase, “wherein the firstand second smoke products are prepared using essentially equivalentprotocols,” as used herein, means that the two smoke products can bevalidly compared. For example, both products can be smoke or bothproducts can be smoke concentrates.

The methods provided herein include methods of identifying a compound intobacco that induces damage of cellular genetic material or modulatescell homeostasis by providing a first tobacco, obtaining smoke or asmoke condensate from the first tobacco, contacting a first populationof cells with the smoke or smoke condensate from the first tobacco,identifying induction of damage of cellular genetic material ormodulation of cell homeostasis in the first population of cells aftercontact with the smoke or smoke condensate from the first tobacco,providing a second tobacco that has been modified to reduce a compoundin the second tobacco, obtaining smoke or a smoke condensate from thesecond tobacco, contacting a second population of cells with the smokeor smoke condensate from the second tobacco, and identifying aninduction of damage of cellular genetic material or modulation of cellhomeostasis in the second population of cells after contact with thesmoke or smoke condensate from the second tobacco, where anidentification of a reduction in the induction of damage of cellulargenetic material or modulation of cell homeostasis in the secondpopulation of cells after contact with the smoke or smoke condensatefrom the second tobacco identifies the compound as one that inducesdamage of cellular genetic material or modulates cell homeostasis.Compounds identified in accordance with the methods provided herein canbe, for example, compounds that induce the double strand DNA breaks,inhibit apoptosis, or inhibit cell proliferation. In some embodiments,the second tobacco can be genetically modified to reduce the expressionof at least one gene that regulates production of the compound.

The compound in tobacco that induces damage of cellular genetic materialor modulates cell homeostasis identified by the methods provided hereincan be a tobacco-derived substance associated with double-strand DNAbreaks (DSBs). The tobacco-derived substance associated with DSBs can bedetected in the context of comparing the harmful potential of twodifferent tobacco or smoke products (as provided herein elsewhere) orcan be detected in an environmental context, such as TS in a businessoffice, train car, or restaurant. The ability to detect the tobaccoderived substance can depend on not only its presence, but also itsconcentration in the “tobacco test composition” (which can be smoke, asmoke concentrate, or, for example, an air sample containing orpotentially containing TS). To that end, useful parameters for assessingthe degree of harmfulness can include, for example, not only the degreeof phosphorylation of H2AX (or accumulation of another DSB marker), butalso the initial rate of DSB accumulation, the period of time requiredto reach a plateau and the degree of phosphorylated DSB at the plateaulevel where a rapid rise in the degree of H2AX phosphorylation, aprotracted period of time to reach a plateau, and a high plateau levelcan be correlated with increased harmful potential (for example, seeFIGS. 14 and 15 and accompanying text). Note that where assay conditionsare relatively prolonged (for example, longer than 55 minutes) it can bedesirable to include, in the assay, a phosphatase inhibitor such ascalyculin A or okadaic acid to inhibit and/or prevent possibledephosphorylation of H2AX molecules.

Also provided herein are methods of identifying a tobacco product thathas a reduced potential to contribute to a tobacco-related disease byproviding a first tobacco product, obtaining smoke or a smoke condensatefrom the first tobacco product, contacting a first population of cellswith the smoke or smoke condensate from the first tobacco product,identifying the presence or absence of an induction of damage ofcellular genetic material or modulation of cell homeostasis in the firstpopulation of cells after contact with the smoke or smoke condensatefrom the first tobacco product, providing a second tobacco product,obtaining smoke or a smoke condensate from the second tobacco product,contacting a second population of cells with the smoke or smokecondensate from the second tobacco product, and identifying the presenceor absence of an induction of damage of cellular genetic material ormodulation of cell homeostasis in the second population of cells aftercontact with the smoke or smoke condensate from the second tobaccoproduct, where an identification of a reduction in the amount or theabsence of an induction of damage of cellular genetic material ormodulation of cell homeostasis in the second population of cells aftercontact with the smoke or smoke condensate from the second tobaccoproduct, as compared to the amount or presence of an induction of damageof cellular genetic material or modulation of cell homeostasisidentified in the first population of cells identifies the secondtobacco product as one that has a reduced potential to contribute to atobacco-related disease. Tobacco products identified as having a reducedpotential to contribute to a tobacco-related disease in accordance withthe methods provided herein can be, for example, tobacco products thatare characterized by a reduced induction of double strand DNA breaks, alower level of inhibition of apoptosis, or a lower level of inhibitionof cell proliferation.

Also provided herein are methods of making a tobacco product that has areduced potential to contribute to a tobacco-related disease byproviding a first tobacco, obtaining smoke or a smoke condensate fromthe first tobacco, contacting a first population of cells with the smokeor smoke condensate from the first tobacco, identifying the presence orabsence or amount of induction of damage of cellular genetic material ormodulation of cell homeostasis in the first population of cells aftercontact with the smoke or smoke condensate from the first tobacco,providing a second tobacco that is genetically modified to reduce theexpression of at least one gene that regulates production of a compoundin the second tobacco, obtaining smoke or a smoke condensate from thesecond tobacco, contacting a second population of cells with the smokeor smoke condensate from the second tobacco, identifying the presence orabsence or amount of induction of damage of cellular genetic material ormodulation of cell homeostasis in the second population of cells aftercontact with the smoke or smoke condensate from the second tobacco,where an identification of a reduction in the presence or amount ofinduction of damage of cellular genetic material or modulation of cellhomeostasis in the second population of cells after contact with thesmoke or smoke condensate from the second tobacco, as compared to thepresence or amount of induction of damage of cellular genetic materialor modulation of cell homeostasis identified in the first cellpopulation identifies the second tobacco as one that has a reducedpotential to contribute to a tobacco-related disease, and incorporationof the second tobacco, which has a reduced potential to contribute to atobacco-related disease, into a tobacco product. Tobacco productsidentified as having a reduced potential to contribute to atobacco-related disease in accordance with the methods provided herein,which are incorporated into a tobacco product, can be, for example,tobacco products that are characterized by a lower induction of doublestrand DNA breaks, lower level of inhibition of apoptosis, lower levelof inhibition of cell proliferation, or reduced level of modulation ofcell homeostasis (e.g., a reduced amount of perturbation of geneexpression; such as reduced amount of expression of genes involved inoncogenesis or a reduced inhibition of genes involved in oxidativerepair as compared to a conventional tobacco product). The section thatfollows describes several methods for identifying a tobacco or tobaccoproducts that modulate cell homeostasis.

Analysis of Changes to Cell Homeostasis: Modulation of Apoptosis

In some embodiments, modulation of cell homeostasis can be identified bydetermining a modulation of apoptosis. Thus, provided herein are methodsof identifying a tobacco that modulates apoptosis by providing atobacco, obtaining a tobacco composition from the tobacco, contacting acell with the tobacco composition, and identifying a modulation ofapoptosis in the cell after contact with the tobacco composition. Alsoprovided herein are methods of identifying a compound in tobacco thatmodulates apoptosis, methods of identifying a tobacco product that has areduced potential to contribute to a tobacco-related disease, andmethods of making a tobacco product that has a reduced potential tocontribute to a tobacco-related disease, in accordance with the methodsof identifying a tobacco or tobacco compound that modulates cellhomeostasis provided herein elsewhere. Also provided herein are methodsof identifying a compound in tobacco that modulates apoptosis, methodsof identifying a tobacco product that has a reduced potential tocontribute to a tobacco-related disease, and methods of making a tobaccoproduct that has a reduced potential to contribute to a tobacco-relateddisease, in conjunction with the methods of identifying a tobacco ortobacco compound that modulates cell proliferation provided herein.

Also provided herein are methods of comparing two or more tobaccoproducts. In some embodiments, a tobacco or tobacco compound thatinduces a lower degree of apoptosis can be characterized as a tobaccothat has a potential to contribute to a tobacco-related disease. In someembodiments, a first tobacco that induces a lower degree of apoptosisthan a second tobacco can be characterized as a tobacco that has anincreased potential to contribute to a tobacco-related disease. In someembodiments, a first tobacco that induces a higher degree of apoptosisthan a second tobacco can be characterized as a tobacco that has areduced potential to contribute to a tobacco-related disease. In someembodiments, a tobacco or tobacco compound that induces a higher degreeof apoptosis can be characterized as a tobacco that has a potential tocontribute to a tobacco-related disease. In some embodiments, a firsttobacco that induces a higher degree of apoptosis than a second tobaccocan be characterized as a tobacco that has an increased potential tocontribute to a tobacco-related disease. In some embodiments, a firsttobacco that induces a lesser degree of apoptosis than a second tobaccocan be characterized as a tobacco that has a reduced potential tocontribute to a tobacco-related disease. In some embodiments, themethods of identifying a tobacco that modulates apoptosis can be used toidentify modified tobacco that modulates apoptosis as provided herein orotherwise known in the art.

Also provided herein are methods of comparing two or more tobaccoproducts. In some embodiments, a tobacco or tobacco compound thatinhibits apoptosis can be characterized as a tobacco that has apotential to contribute to a tobacco-related disease. In someembodiments, upon inducing the same degree of DNA damage (DSBs) a firsttobacco that induces lesser degree of apoptosis than a second tobaccocan be characterized as a tobacco that has an increased potential tocontribute to a tobacco-related disease. In some embodiments, uponinducing the same degree of DNA damage (DSBs) a first tobacco thatinduces lesser degree of apoptosis than a second tobacco can becharacterized as a tobacco that has a reduced potential to contribute toa tobacco-related disease. In some embodiments, a tobacco or tobaccocompound that increases apoptosis can be characterized as a tobacco thathas a potential to contribute to a tobacco-related disease. In someembodiments, a first tobacco that increases apoptosis to a greaterdegree than a second tobacco can be characterized as a tobacco that hasan increased potential to contribute to a tobacco-related disease. Insome embodiments, a first tobacco that increases apoptosis to a lesserdegree than a second tobacco can be characterized as a tobacco that hasa reduced potential to contribute to a tobacco-related disease. In someembodiments, the methods of identifying a tobacco that modulatesapoptosis can be used to identify modified tobacco that modulatesapoptosis as provided herein or otherwise known in the art.

As used herein, a tobacco or tobacco compound that induces a lower orhigher degree of apoptosis refers to a tobacco or tobacco compound thatcauses a cell or cell population to decrease or increase, respectively,apoptosis in that cell or cell population relative to a cell or cellpopulation that is not contacted by the tobacco or tobacco compound. Anyof a variety of methods can be used to determine apoptosis in a cell orcell population, including those provided herein, and other methodsknown in the art.

While not intending to be limited by the following explanation, adecreased degree of apoptosis in cells may result in cells with damagedDNA that can survive and be tumorigenic rather than die and beeliminated. In other cellular functions, extensive apoptosis may inducecompensatory stem cell proliferation and result in tumorigenesis.Accordingly, as contemplated herein an increase or decrease in apoptosiscan lead to a tobacco-related disease.

Also provided herein are methods of comparing two or more tobaccoproducts when the two or more tobacco products induce the same level ofdamage to cells. In some embodiments, a tobacco or tobacco compound thatinhibits apoptosis can be characterized as a tobacco that has apotential to contribute to a tobacco-related disease. In someembodiments, upon inducing the same degree of DNA damage (DSBs) a firsttobacco that induces lesser degree of apoptosis than a second tobaccocan be characterized as a tobacco that has an increased potential tocontribute to a tobacco-related disease. In some embodiments, uponinducing the same degree of DNA damage, a first tobacco that induceslesser degree of apoptosis than a second tobacco can be characterized asa tobacco that has a reduced potential to contribute to atobacco-related disease. In some embodiments, upon inducing the samedegree of DNA damage, a tobacco or tobacco compound that increasesapoptosis can be characterized as a tobacco that has a potential tocontribute to a tobacco-related disease. In some embodiments, uponinducing the same degree of DNA damage, a first tobacco that increasesapoptosis to a greater degree than a second tobacco can be characterizedas a tobacco that has an increased potential to contribute to atobacco-related disease. In some embodiments, upon inducing the samedegree of DNA damage, a first tobacco that increases apoptosis to alesser degree than a second tobacco can be characterized as a tobaccothat has a reduced potential to contribute to a tobacco-related disease.In some embodiments, the methods of identifying a tobacco that modulatesapoptosis can be used to identify modified tobacco that modulatesapoptosis as provided herein or otherwise known in the art.

The methods provided herein can include one or more steps of determiningmodulation of apoptosis. Typically, such methods include assays formodulation of apoptosis in a population of cells. Any of a variety ofmethods known in the art for assaying apoptosis can be used in themethods provided herein. Exemplary known assays include assays foractivation of apoptosis-related proteins, assays for double-strand DNAbreaks, and assays for membrane permeability.

In one exemplary method, modulation of apoptosis can be identified bydetermining caspase activation. Caspases are proteases involved inapoptosis. Activation of caspases can lead to apoptosis in the cell.Accordingly, measurement of activated caspases can be used to identifyapoptosis in cells. Typically, caspases are activated by a cleavagereaction. Thus, activated caspase can be determined by detectingactivated cleaved caspases. For example, caspase activation can beidentified using an antibody or fragment thereof, which binds toactivated caspase but not inactive caspase. There are a number ofcaspases that can be screened in accordance with the methods providedherein, including but not limited to, caspase 1, 3 and 9. In anotherexample, activation of caspase by its catalytic activity can bedetermined. For example, caspase-3 has substrate selectivity for theamino acid sequence Asp-Glu-Val-Asp (DEVD) (SEQ. ID. NO. 1). Afluorogenic indicator such as Ac-DEVD-AMC can be used for fluorometricassay of caspase-3 activity. A variety of caspase activation assays areknown in the art, as exemplified in Gown et al., J. Histochem. Cytochem.(2002) 50:449-54; Iordanov et al., Apoptosis (2005) 10:153-66; andKahlenberg et al., J. Leukoc. Biol. (2004) 76:676-84, all of which arehereby expressly incorporated by reference in their entireties.

In another exemplary method, modulation of apoptosis can be identifiedby determining cleavage of the protein poly(ADP-ribose) polymerase(PARP). Enzymatic cleavage of the PARP occurs uniquely during apoptosis.Activation of caspases results in cleavage of PARP, which producesinactive PARP fragments. One inactive PARP fragment binds DNA andinhibits DNA repair. Thus, cleavage of PARP can be determined using anantibody specific to cleaved PARP fragments. Cleavage of PARP also canbe determined by measuring decrease in PARP activity. PARP catalyzes theNAD-dependent addition of poly(ADP-ribose) to nuclear proteins such ashistone. Thus, in one exemplary assay, incorporation of biotinylatedpoly(ADP-ribose) onto histone proteins can be measured as an indicatorof PARP activity. Methods for determining PARP cleavage are known in theart, as exemplified in Mullen, Methods Mol. Med. (2004) 88:171-81; Yu etal., Science (2002) 297:259-63; and Saldani et al. Eur. J. Histochem.(2001) 45:389-92, all of which are hereby expressly incorporated byreference in their entireties.

In another exemplary method, modulation of apoptosis can be identifiedby determining annexin V binding. Annexin V binds to phosphotidylserineon the cell membrane, a phenomenon that occurs only in cells undergoingapoptosis. In one exemplary assay, fluorescently labeled annexin V canbe added to cells, and presence of the fluorescent marker on the cellsis indicative of annexin binding. In another example, antibodiesspecific for annexin V can be used to detect the presence of annexin Von the cell membrane. This technique is often combined with the use offluorescent dyes that are normally not able to penetrate the cellmembrane unless it is damaged these include dyes such as propidiumiodide and acridine orange. Methods for determining annexin V bindingare known in the art, as exemplified in U.S. Pat. No. 5,767,247, Vermeset al., J. Immunol. Methods (1995) 184:39-51; Wilkins et al., Cytometry(2002) 48:14-9; and Peng et al., Chin. Med. Sci. J. (2002) 17:17-21, allof which are hereby expressly incorporated by reference in theirentireties.

In another exemplary method, modulation of apoptosis can be identifiedby determining chromatin condensation. Chromatin condensation is awell-established indicator of apoptosis. Chromatin condensation can bedetected by a variety of methods, for example, detection by decreasedforward angle light scatter or decreased right angle light scatter, anddetection by presence of a specific dye such as Hoechst 33342. Methodsfor determining chromatin condensation are known in the art, asexemplified in Tounekti et al., Exp. Cell Res. (1995) 217:506-16 andDobrucki et al., Micron (2001) 32:645-52, all of which are herebyexpressly incorporated by reference in their entireties.

In another exemplary method, modulation of apoptosis can be identifiedby determining an increase sensitivity of chromatin in cells to acid orheat-induced denaturation. Sensitivity of chromatin in cells can be amarker of apoptosis. Chromatin sensitivity to acid or heat-induceddenaturation can be detected by a variety of methods known in the art,such as detecting the altered binding of the metachromatic dye acridineorange. Methods for assaying chromatin sensitivity to denaturation areknown in the art, as exemplified in Frankfurt et al., (1996) Exp. CellRes. 226:387-397, Frankfurt et al., (2001) J. Histochem. Cytochem.49:369-378, Frankfurt et al., (2001) J. Immunol. Methods. 253: 133-144,Groos et al., (2003) Anat. Rec. 272A:503-513, Zamzani et al., (1999)Nature 401:127-128, and Allera et al., (1997) J. Biol. Chem.272:10817-10822, all of which are hereby expressly incorporated byreference in their entireties.

In another exemplary method, modulation of apoptosis can be identifiedby determining fractional DNA content. Under appropriate conditions,small molecular weight DNA fragments occurring as the result of theapoptotic process can be removed from cells, resulting in cells withdecreased DNA content. Assays can be used to detect cells with decreased(fractional) DNA content by using, for example, DNA dyes in flowcytometry according to known methods. Methods for assaying fractionalDNA content are known in the art, as exemplified in Mazur et al., Hum.Exp. Toxicol. (2002) 21:335-41 and Gorczyca, Endocrine-Related Cancer(1999) 6:17-19, all of which are hereby expressly incorporated byreference in their entireties.

In another exemplary method, modulation of apoptosis can be identifiedby determining TUNEL assay, as discussed herein elsewhere. TUNEL assaycan detect DNA strand breaks occurring following activation of anapoptosis-specific nuclease. Incorporation of labeled nucleotides at thesite of the double-strand breaks can be detected by, for example,binding of antibodies or other molecules (biotin-avidin) carrying afluorescent tag.

An exemplary assay for cell apoptosis determination is provided inExample 1 for caspase-3 activation measurement. Briefly, cells weretreated with smoke (i.e., A549) or smoke condensate (i.e., NHBE) andfixed as described above, then rinsed twice in PBS and immersed in 0.2%Triton X-100 (Sigma) in a solution of 1% (w/v) bovine serum albumin(BSA; Sigma) in PBS for 30 min to suppress non specific antibodybinding. The cells were then incubated in 100 μl volume of 1% BSAcontaining 1:100 dilution of anti-cleaved (activated) caspase-3 rabbitpolyclonal Ab (Cell Signaling Technology, Beverly, Mass.) overnight at4° C., washed twice with PBS and incubated with 1:30 dilutedFITC-conjugated F(ab′)2 fragment of swine anti-rabbit immunoglobulin(DAKO, Carpinteria, Calif.) for 30 min in room temperature in the dark.The cells were then counterstained with 1 μg/ml4,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, Oreg.) inPBS for 5 min. Each experiment was performed with an IgG control inwhich cells were labeled only with secondary antibody, FITC-conjugatedF(ab′)2 fragment of goat anti-mouse immunoglobulins, without primaryantibody incubation to estimate the extent of nonspecific binding of thesecondary antibody to the cells. The following section describes severalassays that can be used to evaluate the ability of a tobacco or atobacco product to modulate cell proliferation.

Analysis of Changes to Cell Homeostasis: Modulation of CellProliferation

In some embodiments, modulation of cell homeostasis can be identified bydetermining modulation of cell proliferation. Thus, provided herein aremethods of identifying a tobacco that modulates cell proliferation byproviding a tobacco, obtaining a tobacco composition from the tobacco,contacting a cell with the tobacco composition, and identifying amodulation of cell proliferation in the cell after contact with thetobacco composition. Also provided herein are methods of identifying acompound in tobacco that modulates cell proliferation, methods ofidentifying a tobacco product that has a reduced potential to contributeto a tobacco-related disease, and methods of making a tobacco productthat has a reduced potential to contribute to a tobacco-related disease,in accordance with the methods of identifying a tobacco or tobaccocompound that modulates cell homeostasis provided herein elsewhere. Alsoprovided herein are methods of identifying a compound in tobacco thatmodulates cell proliferation, methods of identifying a tobacco productthat has a reduced potential to contribute to a tobacco-related disease,and methods of making a tobacco product that has a reduced potential tocontribute to a tobacco-related disease, in conjunction with the methodsof identifying a tobacco or tobacco compound that modulates cellproliferation provided herein.

Also provided herein are methods of comparing two or more tobaccoproducts. In some embodiments, a tobacco or tobacco compound thatinhibits cell proliferation can be characterized as a tobacco that has apotential to contribute to a tobacco-related disease. In someembodiments, a first tobacco that inhibits cell proliferation to agreater degree than a second tobacco can be characterized as a tobaccothat has an increased potential to contribute to a tobacco-relateddisease. In some embodiments, a first tobacco that inhibits cellproliferation to a lesser degree than a second tobacco can becharacterized as a tobacco that has a reduced potential to contribute toa tobacco-related disease. In some embodiments, a tobacco or tobaccocompound that increases cell proliferation can be characterized as atobacco that has a potential to contribute to a tobacco-related disease.In some embodiments, a first tobacco that increases cell proliferationto a greater degree than a second tobacco can be characterized as atobacco that has an increased potential to contribute to atobacco-related disease. In some embodiments, a first tobacco thatincreases cell proliferation to a lesser degree than a second tobaccocan be characterized as a tobacco that has a reduced potential tocontribute to a tobacco-related disease. In some embodiments, themethods of identifying a tobacco that modulates cell proliferation canbe used to identify modified tobacco that modulates cell proliferationas provided herein or otherwise known in the art.

As used herein, a tobacco or tobacco compound that inhibits or increasescell proliferation refers to a tobacco or tobacco compound that causes acell or cell population to proliferate at a decreased or increased rate,respectively, relative to a cell or cell population that is notcontacted by the tobacco or tobacco compound. Any of a variety ofmethods can be used to determine cell proliferation in a cell or cellpopulation, including those provided herein, and other methods known inthe art.

Any of a variety of assays can be used that monitor alterations to theviability and growth potential of cells in vitro when challenged byexposure to a vast array of insults (e.g., ionizing radiation,ultraviolet radiation, drugs, toxins, carcinogens, CS, CSC, TPM,viruses, chemicals, free radicals, pollution, and the like). Assays thatcan be used in the methods provided herein can include assays thatmonitor proliferative rates (cell proliferation assays) and assays thatmonitor survivability and proliferation with time (e.g., clonogenicsurvival assay).

In one example, clonogenic survival can be monitored. The clonogenicsurvival assay can be used to study the ability of specific agents toimpact the proliferation of cells. This assay is frequently employed incancer research laboratories to determine the effect, if any, of a rangeof substances (e.g., drugs, radiation, chemicals, organic mixtures,etc), on the proliferation of tumor cells. The term “clonogenic” refersto the fact that these cells are clones of one another. Any of a varietyof cell types can be used in such experiments. The cells used typicallycome from established cell lines, which have been well-studied and whosegeneral characteristics are known. Typically, a clonogenic survivalassay has four major steps: (1) inoculating cells into culture dishesand incubate the cells (e.g., 24-48 hours); (2) upon the cells reachingthe logarithmic phase of growth, the treating the cells with a tobaccocomposition (e.g., contacting the cells with freshly prepared anddiluted CS for different periods of time); (3) allowing the cells torecover for a set number of hours (e.g., up to 24 hours), then treatingthe cells and allowing the cells to grow further (e.g., trypsinizing thecells, replating the cells at specific dilutions, and allowing the cellsto grow for 5-7 days); and (4) fixing, staining and counting the cells.Experimental specifics such as time of incubation and growth, number ofcells to use for plating, and the like, can be readily determined by oneskilled in the art according to the type of cell used. Typically, thenumber of surviving colonies of 25-50 cells is representative of thepercentage of cells that survived the treatment. A graphicalrepresentation of survival versus exposure time to a tobacco compositioncan then be generated. The surviving fraction can be determined bydividing the number of colonies in the dish by the number of thecolonies in the control (non-treated) dish.

In addition to clonogenic assays, any of a variety of cell proliferationassays can be used to monitor an increase or decrease in proliferativecapacity and which can be used in context with exposure to a tobaccocomposition such as CS, CSC and/or TPMs.

In one example of cell proliferation assays, intake and conversion of adye can be an indicator of cell proliferation. One example of such anassay is a resazurin-based assay. Resazurin is a redox dye which is notfluorescent, but upon reduction by metabolically active cells, isconverted into a highly fluorescent product (resorufin). Living cellscan readily reduce this non-toxic reagent and the resulting increase influorescence intensity is monitored using a fluorescencespectrophotometer or plate reader. Exemplary commercially availableassays include alamarBlue™ reagent from BioSource International,Camarillo Calif.

Another example of dye intake and conversion-based cell proliferationassays is a tetrazolium salt-based assay. The tetrazolium salt assay isa colorimetric assay is based on the conversion of a tetrazolium salt(MTT, WST, or other) to formazan, a purple dye. This cellular reductionreaction involves the pyridine nucleotide cofactors NADH/NADPH and isonly catalyzed by living cells. The formazan product has a low aqueoussolubility and is present as purple crystals. Dissolving the resultingformazan with a solubilization buffer permits the convenientquantification of product formation. The intensity of the product coloris directly proportional to the number of living cells in the culture.Exemplary commercially available assays include Quick Cell ProliferationAssay Kit from BioVision Inc., Mountain View, Calif.

In another example of cell proliferation assays, cells can be monitoredfor plasma membrane damage. Plasma membrane damage-based assays can beused to monitor cell death or cytotoxicity. Typical assays quantitatemolecules released from damaged cells such as adenylate kinase andlactate dehydrogenase. Exemplary commercially available assays includeLDH-Cytotoxicity Assay Kit from BioVision Inc., Mountain View, Calif.

In another example of cell proliferation assays, cells can be monitoredfor dye exclusion/dye uptake assays. Dye exclusion/uptake assaysdistinguish live from dead cells based on dyes which specifically staineither live or dead cells. Exemplary commercially available assaysinclude trypan blue dye exclusion, Live-Dye™ (a cell-permeable greenfluorescent dye that stains live cells) from BioVision Inc., MountainView, Calif.

In another example of cell proliferation assays, cells can be monitoredfor ATP and ADP levels. ATP and ADP level-based assays utilize thephenomenon that increased levels of ATP and decreased levels of ADP havebeen recognized in proliferating cells. Exemplary commercially availableassays include ApoSENSOR™ Cell Viability Assay Kit from MBLInternational, Woburn Mass.

In another example of cell proliferation assays, cells can be monitoredfor protein or DNA levels in the cells. Cell proliferation is associatedwith increased protein and DNA synthesis. DNA quantitation-based assayscan use, for example, [3H]-thymidine incorporation, the fluorescence ofa DNA-dye complex from lysed cells, or other known markers of DNAsynthesis. Similarly, protein synthesis can be monitored forincorporation of labeled amino acids into the proteins. Exemplarycommercially available assays include Quantos™ Cell Proliferation AssayKit from Stratagene, La Jolla, Calif.

Example 3 below provides one non-limiting specific example of theclonogenic survival assay methods provided herein. Variations of theassay method used in terms of materials, assay times, instrumentationand protocols would be apparent to the skilled artisan.

Example 3

A clonogenic survival assay was used to study the ability of tobaccosand tobacco products to impact the proliferation of cells. Theexperiment involves four major steps: (1) inoculate cells into culturedishes and incubate for 24-48 hours; (2) upon reaching the logarithmicphase of growth, the treatment is applied; the treatment in this case isfreshly prepared and diluted CS for increasing periods of time; (3) thecells are then allowed to recover for a set number of hours (up to 24),then the cells are trypsinized, replated at specific dilutions, andallowed to continue growing for 5-7 days; the number of cells useddepends largely on the plating efficiency of the cell line and must bedetermined empirically prior to the experiment; and (4) at theconclusion of the experiment, the cells are fixed, stained, and counted.The primary measure is to count surviving colonies of 25-50 cells whichis presented as the percentage of cells which survived the treatment. Agraphical representation of survival versus exposure time to CS is thengenerated. The surviving fraction is determined by dividing the numberof colonies in the dish by the number of the colonies in the control(non-treated) dish.

A549 cells were exposed to smoke as described above. Following smokeexposure the medium is aspirated and the cells rinsed refed with 37° C.BEGM and placed in a 37° C., 5% CO2 humidified incubator for two tothree hours. The cells are harvested by trypsinization with trypsin-EDTA(0.25% trypsin-0.38 mg/ml EDTA, Invitrogen). Cells are centrifuged at260×g for 8 min. Cell pellets are resuspended in 1 ml of Ham's F-12Kmedium, 10% FBS (complete medium) per pellet and counted. Cells areserially diluted so that the mock treated have ˜65 cells per well andsmoke treated have ˜300 cells per well when seeded onto 96-well flatbottom tissue culture plates; one plate per condition. The plates areincubated for five days in a 37° C., 5% CO2 humidified incubator. Thecolonies of cells are fixed with 5% formaldehyde/PBS and colored with0.8% crystal violet solution for visualization. The colonies are countedwith the aid of a macroscopic dissecting microscope. The cloningefficiency results are expressed in relation to the mock exposed cells.Unless otherwise indicated, each bar in the graphs represents threereplicate data points per experiment.

A549 cells were exposed to whole smoke from IM16 or Marlboro® cigarettesfor various lengths of time after which clonogenic assays wereperformed. FIG. 25 is a summary of multiple experiments. The numbers inparentheses indicate the number of experiments represented by each bar.The industry monitor reference cigarette IM16 shows an effect onviability essentially identical to that of the Marlboro® cigarettes. Inboth cases there was a linear decrease in cell viability with increasingsmoke exposure.

In one set of experiments, A549 cells were exposed to smoke from variouscigarettes for 20 min and clonogenic assays were performed. IM16, Omni®,Marlboro®, Quest 1 ®, or Quest 3® brand cigarettes were compared. Eachgraph of FIG. 26 represents a separate experiment. The assaydistinguishes between the cigarettes, with Quest 3® treatment having theleast impact on cell viability and IM16 having the greatest. An overallranking of the cigarettes in terms of impact on viability can be seen:Quest 3®<Quest 1® and Omni®<Marlboro®<IM16. Thus, the tobacco productscontaining modified tobacco (i.e., Omni®, Quest 1®, and Quest 3® had thean impact on cell viability that was significantly less than a referencetobacco product (i.e., IM16) and a conventional, commercially available,traditional tobacco product (i.e., Marlboro®). Accordingly, the modifiedtobacco products Omni®, Quest 1®, and Quest 3® have a reduced potentialto contribute to a tobacco related disease (i.e., Omni®, Quest 1®, andQuest 3® are reduced risk tobacco products) according to the clonogenicassay.

In a next set of experiments, the mitigation of the effect of wholesmoke on cell viability by the presence of NAC was evaluated. A549 cellswere exposed to 20 min IM16 smoke in the presence of variousconcentrations of the free radical scavenger N-acetylcysteine (NAC) andthe clonogenic assay performed. NAC protected the viability of the cellsin a dose-dependent manner. FIG. 27 shows the increasing degree ofproliferation resulting from increasing concentrations of NAC.

In another series of experiments, the effect of NAC on the viability ofcells contacted with whole smoke from different cigarettes wasevaluated. A549 cells were exposed to smoke from various cigarettes for20 min in the presence or absence of 25 mM NAC and the clonogenic assayperformed. IM16, Omni®, and Quest 3® cigarettes were compared. NACcompletely protected the cells exposed to Quest 3® smoke, and partiallyprotected cells exposed to Omni® or IM16 (FIG. 28). Again, these datashow that tobacco products containing modified tobacco (i.e., Omni® andQuest 3®) had the an impact on cell viability that was significantlyless than a reference tobacco product (i.e., IM16). Accordingly, themodified tobacco products Omni® and Quest 3® have a reduced potential tocontribute to a tobacco related disease (i.e., Omni® and Quest 3® arereduced risk tobacco products).

In yet another series of experiments, the effect of NAC on cell deathcaused by the VAPOR phase of smoke from different cigarettes wasevaluated. A549 cells were exposed to the vapor phase of smoke for 20min by inserting a Cambridge filter pad immediately after the cigarettein the smoking apparatus so as to filter out the particulate matter(“tar”) and leave only the vapor phase. Three different cigarettes wereused: IM16, Quest 1® and Quest 3®. Cells were exposed in the presence orabsence of 25 mM NAC. The clonogenic assay was subsequently performed.

The vapor phase of all cigarettes showed less effect on cell viabilitythan the corresponding whole smoke exposure, with Quest 3® exhibitingalmost no effect (FIG. 29). The effect of various cigarettemodifications on vapor phase toxicity can thus be selectively monitored.In all vapor phase exposures, the presence of the free radical scavengerNAC protected the cells against viability loss. These experimentsprovide more evidence that the tobacco products containing modifiedtobacco (i.e., Quest 1®, and Quest 3® had an impact on cell viabilitythat was significantly less than a reference tobacco product (i.e.,IM16) and, thus, Quest 1®, and Quest 3® have a reduced potential tocontribute to a tobacco related disease (i.e., Quest 1® and Quest 3® arereduced risk tobacco products).

Filter Comparison

Clongenic assays also were applied to tests of several filters attachedto different tobaccos. Filters and tobacco were obtained from: (1) theindustry standard reference tobacco IM16 (Philip Morris® USA); (2)reduced risk cigarette Omni® (Vector Tobacco Ltd.); (3) reduced riskcigarette Quest 1® (Vector Tobacco Ltd.), and (4) reduced risk cigaretteQuest 3® (Vector Tobacco Ltd.). A549 cells were exposed to mocktreatment (control) and cigarette smoke substantially as provided in theabove smoke treatment description.

Numerous combinations of tobacco and filters from IM16, Omni®, Quest 1®and Quest 3® were tested, and the cloning efficiency relative to mock ispresented in FIGS. 45-47. FIG. 45 shows clonogenic results for each ofIM16, Omni®, and Quest 3® with the cigarette in tact, and the filter cutand then reattached to the same tobacco rod. FIG. 45 further showsclonogenic results for Omni® and Quest 3® filters attached to IM16tobacco rods, and IM16 filters attached to Omni® and Quest 3® tobaccorods. The results show that while there was some variation in cloningefficiency when filters were cut and reattached to the same tobacco rod,Omni® and Quest 3® filters attached to IM16 tobacco rods providedincreased cloning efficiency, while the IM16 filter attached to theQuest 3® tobacco rod provided decreased cloning efficiency. Theseresults show that different filters attached to the same tobacco rodhave different influences on cloning efficiency, where the cloningefficiencies are inversely related to the expected levels of riskattributed to the tobacco product (IM16 is highest expected risk and hasthe lowest cloning efficiencies, while Quest 3® is lowest expected riskand has the highest cloning efficiencies). Similar experiments wererepeated: (1) where IM16, Quest 1® and Quest 3® tobaccos and filterswere exchanged and compared (FIG. 46) and (2) where cloning efficiencywas tested at 7 days (FIG. 47). The results in FIGS. 46 and 47 arecomparable to those of FIG. 45 and again reflect inverse relationshipbetween the expected levels of risk attributed to the tobacco productand cloning efficiency. The following section describes severalepidemiological approaches to determine the potential of a tobacco or atobacco product to contribute to a tobacco related disease.

Analysis of Changes in Cell Homeostasis: Modulation of the Transcriptomeor Proteome

Provided herein are methods for identifying a tobacco that modulatescell homeostasis by providing a tobacco, obtaining a tobacco compositionfrom the tobacco, contacting a cell with the tobacco composition, andidentifying any modulation of the cell transcriptome or proteome aftercontact with the tobacco composition. In some embodiments, the methodsprovided herein can monitor induction of expression of a gene that issilent during homeostasis or repression a gene that is active duringhomeostasis. In some embodiments, the tobacco composition can be smokeor smoke condensate.

The methods provided herein can be used to characterize a first and asecond tobacco product by providing the first and second tobaccoproducts, obtaining a first and second tobacco composition from thefirst and second tobacco products, respectively, contacting a first cellwith the first tobacco composition and contacting the second cell withthe second tobacco composition, and identifying one or more attributesof the transcriptome or proteome of the contacted cells. Differenttobacco products can contain different levels of carcinogens that caninduce various types of changes to mRNA or protein levels, ormodifications of mRNA or protein molecules. Comparison of attributes ofcells contacted with different tobacco compositions can be performed inthe methods provided herein, and such attributes include, but are notlimited to, differential levels of mRNA, differential levels of proteinand changes to the post-tranlsational protein modifications.Accordingly, the methods provided herein can be used to compare two ormore tobacco products by assay methods including assay for differentiallevels of mRNA, differential levels of protein, and changes topost-tranlsational protein modification. Exemplary assay methods includemicroarray assays, qRT-PCR assays, Western blota assays, and ELISAassays.

By one approach, for example, CS is generated using a smoking machinefrom a first tobacco modified product, e.g., a product containingtobacco that has been genetically modified to have a reduced amount of acompound. A first population of NHBE cells is contacted with said CSobtained from the modified tobacco product, and the cells contacted withCS are assayed for mRNA or protein levels. A second population of NHBEcells is then contacted with CS generated from an unmodified orreference tobacco product. The second population of cells contacted withCS is then assayed for mRNA or protein levels. A comparison of the dataobtained from the analysis of the first and second tobacco products willreveal that the difference in mRNA or protein levels caused by themodified tobacco product relative to the unmodified tobacco product. Bythis approach, one can effectively identify the contribution ofindividual components of a tobacco product to mRNA or protein levels, orother assay conditions provided herein or otherwise known in the art.These methods can thereby be used to identify the contribution ofindividual components of a tobacco product to a tobacco-related disease.This approach can be used to develop tobacco products that are lesslikely to contribute to a tobacco-related disease and reduced risktobacco products identified by these methods are embodiments providedherein. Further, tobacco products prepared by these approaches can beprepared according to good manufacturing processes (GMP) (e.g., suitablefor or accepted by a governmental regulatory body, such as the FederalDrug Administration (FDA), and containers that house said tobaccoproducts can comprise a label or other indicia, with or withoutstructure-function indicia, which reflects approval of said tobaccoproduct from said regulatory body.

In a first series of experiments, the influence of cigarette smokecondensates (CSC) from two different tobacco products (cigarettes) onthe gene expression of NHBE cells was examined. In a second set ofexperiments, the influence of cigarette smoke (CS) generated from onetobacco product (a cigarette) on the gene expression of NHBE cells wasexamined. Although NHBE cells are preferred for the methods describedherein, other cells of the mouth, oral cavity, trachea, and lungs,either normal or immortalized cell lines (e.g., human bronchial cells(e.g., BEP2D or 16HBE140 cells), human bronchial epithelial cells (e.g.,HBEC cells, 1198, or 1170-I cells), normal human bronchial epithelialcells, BEAS cells (e.g., BEAS-2B), NCI-H292 cells, non-small cell lungcancer (NSCLC) cells or human alveolar cells (e.g., H460, H1792,SK-MES-1, Calu, H292, H157, H1944, H596, H522, A549, and H226) tonguecells (e.g., CAL 27), and mouth cells (e.g., Ueda-1)) can be used.Accordingly, several embodiments concern methods of identifying one ormore genes present in human cells of the mouth, tongue, oral cavity,trachea, or lungs (e.g., NHBE cells) that are modulated by exposure toCS, CSC, TS, TSC or TPM.

In some embodiments, the methods include providing a first population ofisolated human cells of the mouth, tongue, oral cavity, or lungs (e.g.,NHBE cells), contacting the cells with a CS, CSC, TS, TSC or TPM from afirst tobacco product (e.g., a cigarette) in an amount and for a timesufficient to modulate expression or modification of one or more genesor gene products, and identifying the gene that is modulated or themodified gene product (e.g., phosphorylated) or the level or amount ofgene expression or modification. The identification of a gene that ismodulated or modified gene product or the level or amount of geneexpression or presence or absence of a modification on a gene productcan be accomplished using any technique available that analyzestranscription (e.g., microarray, genechip, oligonucleotide array, anamplification technique, qRT-PCR, or hybridization), protein production(e.g., ELISA, Western blot, or other antibody detection techniques), ormodifications of proteins (e.g., oxidation or phosphorylation, such asdetection methods that employ anti-phospho-tyrosine antibodies).Additionally, the appearance or disappearance of metabolites associatedwith genes that are modulated in response to exposure to CS, CSC, TS,TSC or TPM can also be monitored (e.g., cysteine, glutathione, fragmentsof proteins or lipids or fatty acids) using techniques that areavailable.

In some embodiments, the pattern and/or level of gene expression or geneproduct modification of a control population (e.g., a second populationof isolated human cells of the mouth, tongue, oral cavity, or lungs(e.g., NHBE cells)), is compared to the level of expression or geneproduct modification in the first population of isolated cells. By thisapproach, preferably using the same type of cells for each of the twopopulations, a first population is contacted with a CS, CSC, TS, TSC orTPM and the second population of isolated cells is not. In this manner,the second population of isolated cells is a control population, whichwill exhibit the baseline pattern or level or amount of gene expressionor gene product modification (homeostasis). Data generated from thefirst or second population of isolated cells before or after exposure toCS, CSC, TS, TSC, TPM or air (control) can be recorded on a computerreadable media and databases containing this information can be used toidentify a gene that is modulated in response to contact with a CS, CSC,TS, TSC or TPM or to investigate the gene expression pathways that leadto a particular tobacco-related disease.

In some embodiments, a second tobacco product (e.g., a cigarette) iscompared to a first tobacco product (e.g., a cigarette) using theanalysis above. That is, for example, a first population of isolatedhuman cells of the mouth, tongue, oral cavity, or lungs (e.g., NHBEcells), is contacted with a CS, CSC, TS, TSC or TPM from a first tobaccoproduct (e.g., a cigarette) in an amount and for a time sufficient tomodulate expression of one or more genes or to modify a gene product,and identification of a gene that is modulated or modified gene product(e.g., phosphorylated) or the level or amount of gene expression ormodification can be determined using any technique available thatanalyzes transcription (e.g., qRT-PCR or hybridization), proteinproduction (e.g., ELISA, Western blot, or other antibody detectiontechniques), modifications of proteins (e.g., oxidation orphosphorylation), or the appearance or disappearance of metabolitesassociated with genes that are modulated in response to exposure to CS,CSC, TS, TSC or TPM (e.g., cysteine, glutathione, fragments of proteinsor lipids or fatty acids). A second population of isolated human cellsof the mouth, tongue, oral cavity, or lungs (e.g., NHBE cells),preferably the same type of cell as used in the analysis of the firsttobacco product, is also contacted with a CS, CSC, TS, TSC or TPM from asecond tobacco product (e.g., a cigarette) in an amount and for a timesufficient to modulate expression of one or more genes or to modify agene product. Identification of a gene that is modulated or modifiedgene product (e.g., phosphorylated) or the level or amount of geneexpression or modification can also be accomplished using any techniqueavailable that analyzes transcription (e.g., qRT-PCR or hybridization),protein production (e.g., ELISA, Western blot, or other antibodydetection techniques), modifications of proteins (e.g., oxidation orphosphorylation), or the appearance or disappearance of metabolitesassociated with genes that are modulated in response to exposure to CS,CSC, TS, TSC or TPM (e.g., cysteine, glutathione, fragments of proteinsor lipids or fatty acids).

The data obtained from the analysis of the first tobacco product can becompared to the data obtained from the analysis of the second tobaccoproduct so as to identify, for example, a gene(s) that is induced inresponse to exposure to the first tobacco product but not the secondtobacco product or vice versa. Additionally, the comparison will revealthat the level of expression of one or more genes induced by bothtobacco products differs with respect to the two tobacco products orthat the first product has more, less, or no modification of aparticular gene product (e.g., phosphorylation), as compared to thesecond tobacco product or vice versa. These data (e.g., the types ofgenes expressed, the amount of expression, and modification) allow oneto develop a profile for each tobacco product analyzed (in this exampleonly two products are being compared but a plurality of products can becompared using the same approach). These tobacco product profiles can berecorded on a computer readable media and databases containing thisinformation can be created. Many of the genes that are expressed, theamount of expression, and/or modification can be associated withmolecular events that contribute to a tobacco related disease. Byanalyzing the differences between the tobacco products analyzed, (e.g.,the types of genes expressed, the amount of expression, andmodification), one can identify a tobacco product that has lesspotential to contribute to a tobacco related disease or that, forexample, a first tobacco product has a reduced risk to contribute to atobacco-related disease, as compared to a second tobacco product or viceversa. Thus, reduced risk tobacco products identified by the approachesdescribed herein are embodiments of the invention.

More embodiments concern methods to identify components of CS, CSC, TS,TSC or TPM that modulate the expression of a gene that contributes to atobacco-related disease. In one embodiment, the pattern or level of geneexpression or modification of a gene product in cells of the mouth, oralcavity, trachea, or lung (e.g., NHBE cells) that are exposed to a firsttobacco product that lacks a component associated with a tobacco-relateddisease (e.g., nicotine) is compared to a second tobacco product(preferably of the same type of tobacco as the first tobacco product)that contains the component (e.g., nicotine) and the impact on the typesof genes expressed, the amount of expression, and modification of geneproducts is analyzed (e.g., microarray analysis, Western blot, ELISA,and/or qRT-PCR). By this approach, the genes or modifications of a geneproduct, which are modulated as a result of the presence or absence ofthe component (e.g., nicotine), can be identified. Because many of thesemodulated genes or modifications of gene products will be associatedwith molecular events that contribute to a tobacco-related disease, onecan rapidly identify whether the presence or absence of a particularcomponent in a tobacco product elevate the risk of acquiring aparticular tobacco-related disease. Once a component that contributes toa tobacco-related disease has been identified using the approachesdescribed herein, one can use various techniques to remove thiscomponent from tobacco (e.g., genetic modification, chemical treatment,or adjustments in the harvesting, curing, or processing of the tobacco)and thereby develop reduced risk tobacco products (e.g., cigarettes).Thus, reduced risk tobacco products identified by these approaches areembodiments of the invention.

Many embodiments described herein employ normal human bronchial cells(NHBE cells) that are maintained in culture. Although NHBE cells arepreferred for the methods described herein, it should be understood thatmany other cells that are typically contacted with tobacco or tobaccosmoke during the process of smoking (e.g., lung cells, bronchial cells,cells of the mouth, pharynx, larynx, and tongue) can also be used.Additionally, many immortal cell lines can be used with the methodsdescribed herein. Preferred cells for use with the embodied approachesinclude, but are not limited to, human bronchial cells (e.g., BEP2D or16HBE140 cells), human bronchial epithelial cells (e.g., HBEC cells,1198, or 1170-I cells), normal human bronchial epithelial cells, BEAScells (e.g., BEAS-2B), NCI-H292 cells, non-small cell lung cancer(NSCLC) cells or human alveolar cells (e.g., H460, H1792, SK-MES-1,Calu, H292, H157, H1944, H596, H522, A549, and H226), tongue cells(e.g., CAL 27), and mouth cells (e.g., Ueda-1)). Many of such culturesare available commercially or through a public repository (e.g., ATCC).Further, several techniques exist that allow for one to generate primarycultures of said cells and these primary cultures can be used with themethods described herein.

Example 4 Treatment of NHBE Cells with CSCs

The tobacco smoke condensates were prepared as follows. Smoke wasgenerated from two commercially available nationally sold brands ofAmerican cigarettes (Brand A and Brand B) using an INBIFO-Condor smokingmachine under Federal Trade Commission (FTC) smoking parameters (2.0second puff duration, 35 milliliter puff every 60 seconds). Both brandsof cigarettes were non-menthol, full-flavor types of American-blendedcigarettes with averaged FTC measured values of 13.2 mg tar/0.88 mgnicotine (Brand A), and 14.5 mg tar/1.04 mg nicotine (Brand B). Brand Acontains tobacco that has been chemically modified to reduce carcinogens(see U.S. Pat. No. 6,789,548, herein expressly incorporated by referencein its entirety), whereas Brand B contains conventional tobacco. Smokecondensates extracted from these two cigarette brands and designatedCSC-A and CSC-B, respectively, were collected from the smoke via aseries of three cold traps (−10° C., −40° C., and −70° C.) ontoimpingers filled with glass beads. The condensates were dissolved inacetone, which was then removed by rotary evaporation at 35° C. Theresulting cigarette smoke condensates (CSCs) were weighed and dissolvedin dimethylsulfoxide (DMSO) to make stock solutions of each condensateat a concentration of 40 mg/mL, which were stored at −20° C. prior touse.

NHBE (Normal Human Bronchial Epithelial) cells were purchased fromCambrex Corporation, East Rutherford, N.J. The cells were cultured incomplete Bronchial Epithelial Cell Growth Medium (BEGM), prepared bysupplementing Bronchial Epithelial Basal Medium with retinoic acid,epidermal growth factor, epinephrine, transferrin, T3, insulin,hydrocortisone, antimicrobial agents and bovine pituitary extract byaddition of SingleQuots,™ (both purchased from Cambrex Corporation, EastRutherford, N.J.). S9 metabolic fraction from Aroclor 1254-treated ratswas obtained from BioReliance Corporation, Rockville, Md. A 5×concentration of S9 microsomal fraction with cofactors was preparedimmediately before treating the cells, and contained 10% S9 microsomalfraction, 4 mM NADP, 5 mM glucose-6-phosphate, 50 mM phosphate buffer pH8.0, 30 mM KCl, and 10 mM CaCl₂).

Twenty-eight flasks were seeded with 14.6 ml of a 2.52×10⁴ cells/ml cellsuspension and an additional 15.4 ml pre-warmed BEGM were added to eachflask for a final volume of 30 mL/flask. All incubations were at 37° C.in a humidified atmosphere of 5% CO₂ in air. Cells were grown to 40%confluence, at which time the cultures were treated. Four flasks wereused as untreated control cultures. Following medium removal in thesefour control flasks, the cells were re-fed with 30 ml pre-warmed BEGMand their RNA harvested at 0 h (2 flasks) and 20 hr (2 flasks). Theremaining 24 experimental flasks were treated with either CSC-A in thepresence of 2% S9 microsomal fraction, CSC-B in the presence of 2% S9fraction, or 2% S9 microsomal fraction alone. Following medium removal,each flask received 9.0 ml of fresh BEGM, 15.0 mL BEGM containing CSC orvehicle (400 μg/ml of CSC-A or CSC-B and 1% DMSO for the CSC-treatedgroups, 15.0 mL containing 1% DMSO for the S9-only group), and 6 ml of5× S9 fraction for a final concentration of 2% S9 and a final mediavolume of 30 mL. Incubation was carried out under the incubationconditions described above. Duplicate flasks were used for eachtreatment/time point of the experiment (i.e., 2, 4, 8, and 12 h).

The monolayer cultures of NHBE cells were treated in logarithmic phaseof growth for up to 12 hours with CSC-A or CSC-B in the presence of 2%S9 microsomal fraction, or with 2% S9 fraction alone. Cell viabilityafter 12 hours exposure was 84% and 73% for CSC-A and CSC-B treatments,respectively, when compared to untreated cells. RNA was then extractedfrom cells at 2, 4, 8, and 12 hours post-treatment, fluorescentlylabeled and hybridized to genome-scale microarrays, as described in theexamples that follow.

Treatment of NHBE Cells with CS

Two identical and independent smoke exposure experiments using NHBEcells were performed. In both experiments, the cells were exposed tocigarette smoke (CS) or air (“mock-exposed”) for 15 min, after which thecells were re-fed with fresh media and allowed to incubate for either 4h or 24 h (the “washout” period). In preparation for exposure, cellswere seeded into 35 mm Petri dishes (Fisher Scientific, Falcon #35-3001,Pittsburgh, Pa.) at a density of 105 cells/dish. This resulted in nomore than 70% confluence at the time of smoke treatment 48 hours later.

Experiment 1 used cells from a 23-year-old nonsmoking, non-diabetic maledonor purchased from Cambrex Corporation (Walkersville, Md.). A total often Petri dishes were treated: two dishes were mock-exposed with a 4 hwashout, two dishes were CS-exposed with a 4 h washout, three disheswere mock-exposed with a 24 h washout, and three dishes were CS-exposedwith a 24 h washout.

Experiment 2 was performed in an essentially identical manner asExperiment 1, except for the cell donor (a 13-year-old nonsmoking,non-diabetic male, purchased from Cambrex Corporation, Walkersville,Md.), and the number of Petri dishes used for the mock- and CS-exposedsamples with a 24 h washout (two instead of three). This resulted in atotal of eight Petri dishes treated for Experiment 2.

Smoke was generated from a commercially available, nationally sold,non-menthol, full-flavor brand of American filter cigarettes (averagedFTC measured values of 14.5 mg tar/1.04 mg nicotine) using a KC 5 PortSmoker (KC Automation, Richmond, Va.) smoking machine under FederalTrade Commission (FTC) smoking parameters (35±0.3 cc puff volume, onepuff every 60 seconds, 2-second puff duration with none of theventilation holes blocked, using cigarettes which have been equilibratedat 23.9° C.±1.1° C. and 60%±2% relative humidity for a minimum of 24hours and a maximum of 14 days).

Immediately prior to smoke exposure the culture medium was removed fromeach dish and replaced with pre-warmed Dulbecco's Phosphate BufferedSaline (PBS) containing calcium and magnesium (BioSource, Rockville,Md.). The Petri dishes were placed in a smoke exposure chamber (20.6cm×6.7 cm×6.3 cm). Each 35 cc puff was diluted to 500 cc usingcompressed air containing 5% CO2 and then was drawn over the cells withthe aid of a vacuum pump in order to keep a constant flow of smoke overthe cells with minimal accumulation in the exposure chamber. Cigaretteswere smoked to a maximum of seven puffs per cigarette, within 3 mm ofthe filter tip. Mock exposure conditions were identical to smokeconditions without a cigarette placed in the smoking port. Immediatelyafter exposure, the PBS was removed from each dish and replaced withfresh pre-warmed cell culture medium. The Petri dishes were transferredto a 37° C. 5% CO2 incubator and incubated for 4 or 24 hourspost-exposure.

Cells were cultured in complete Bronchial Epithelial Cell Growth Medium,prepared by supplementing Bronchial Epithelial Basal Medium withretinoic acid, epidermal growth factor, epinephrine, transferrin, T3,insulin, hydrocortisone, antimicrobial agents and bovine pituitaryextract by addition of SingleQuots,™ (Cambrex Corporation, Walkersville,Md.). All incubations were at 37° C. in a humidified atmosphere of 5%CO2 in air. All cells were used before their fifth passage, althoughNHBE cells can be used up to 10 passages or more in the methodsdescribed herein.

Once the cells are contacted with a CS, CSC, TS, TSC or TPM, an approachto analyze the genes that are modulated in response to the exposure isemployed. In some embodiments, the identification of at least one genethat is modulated by exposure to CS, CSC, TS, TSC or TPM is accomplishedusing an array technology, an oligonucleotide array technology, agenechip technology, any type of hybridization or blot, PCR, QRT-PCR,another amplification technology or protein detection methodologies,such as antibody detection methods, ELISA and Western blot. In someembodiments, the identification is made by observing a modulation(up-regulation or down-regulation) in the level or activity of an mRNAand/or a protein. In some embodiments, the modulation is seen as anincrease in mRNA or protein production. In other embodiments, themodulation is seen as a decrease in mRNA or protein production. In someembodiments, the modulation is identified as being statisticallyrelevant. In some embodiments, the presence or absence of a modificationof a gene product (e.g., phosphorylation, acylation, or cleavage of apeptide) or the presence or absence of a metabolite (e.g., cysteine orglutathione) is analyzed. In still more embodiments the modulation,modification, metabolite or amounts thereof are recorded on a computerreadable medium (e.g., disc drive, floppy, CD-ROM, DVD-ROM, zip disc,memory cache, and the like). Accordingly, specific genes or patterns ofgenes and modified gene products that appear in response to exposure toCS, CSC, TS, TSC or TPM can be identified, recorded on a computerreadable medium and this data can be used to generate a profile for eachproduct tested.

In the example that follows, approaches that were used to analyze thepattern and level of expression of genes from NHBE cells exposed to atobacco smoke condensate (CSC) from two different tobacco products aredescribed.

Example 5 Isolation of RNA from CSC-Treated Cells and Production of cDNA

After NHBE cells were exposed to the cigarette smoke condensates (CSC-Aand CSC-B), as explained in Example 4, RNA was prepared by harvestingcells for total RNA extraction after 0 (untreated), 2, 4, 8, and 12hours of treatment. The medium was aspirated and the flasks were rinsedtwice with pre-warmed 15 mL Dulbecco's Phosphate Buffered Saline. Afterthe second rinse, 5.0 mL of cold TRIzol® (Invitrogen Corp., Carlsbad,Calif.) were added to cover the cells in each flask. Each flask wasvigorously vortexed for approximately one minute. The TRIzol® waspipetted up and down over the surface of the flask at least five timesto suspend the cell lysate. The resulting TRIzol®/cell lysate wasallowed to remain in the flask for at least 10 minutes at roomtemperature after which it was transferred to microfuge tubes andextracted with 0.2 ml chloroform per 1.0 ml TRIzol/cell lysate. Thetubes were capped and shaken vigorously to initiate the RNA extraction,and centrifuged at >15,000×g for two 5-minute spins. Following thesecond 5-minute centrifugation, the aqueous layer was collected (˜500μl) and transferred to a second set of microfuge tubes containing anequal volume of isopropyl alcohol. The samples were centrifuged for 30minutes at >15,000×g. Following centrifugation, most (˜90%) of theliquid was removed from the microfuge tube. The remaining RNA pellet wasfrozen and stored at <−60° C. RNA was resuspended indiethylpyrocarbonate-treated water. RNA integrity was assessed usingcapillary gel electrophoresis (Agilent Technologies, Palo Alto, Calif.)to determine the ratio of 28s:18s rRNA in each sample. cDNA wassynthesized with a direct incorporation of Cy3-dUTP from 2 μg total RNAusing Clontech Powerscript (Clontech, Palo Alto, Calif.) reversetranscriptase. Labeled cDNA was then purified using a Montage 96-wellvacuum system.

Microarray Printing and Processing in CSC Experiments

The microarrays used in experiments involving CSC-treated cells werepurchased from the Oklahoma Medical Research Foundation MicroarrayResearch Facility. Slides were produced using commercially availablelibraries of 70 nucleotide long DNA molecules whose length and sequencespecificity were optimized to reduce the cross-hybridization problemsencountered with cDNA-based microarrays (Human Genome Oligo Set Version2.0, Qiagen, Valencia, Calif.). The microarrays had 21,329 human genesrepresented. The oligonucleotides were derived from the UniGene andRefSeq databases. The RefSeq database is an effort by the NCBI to createa true reference database of genomic information for all genes of knownfunction. For the genes present in this database, information on genefunction, chromosomal location, and reference naming are available. All11,000 human genes of known or suspected function are represented onthese arrays. In addition, most undefined open reading frames wererepresented (approximately 10,000 additional genes). Oligonucleotideswere resuspended at 4004 concentrations in 3×SSC and spotted ontoCorning® UltraGAPS™ amino-silane coated slides, rehydrated with watervapor, snap dried at 90° C., and then covalently fixed to the surface ofthe glass using 300 mJ, 254 nm wavelength ultraviolet radiation. Unboundfree amines on the glass surface were blocked for 15 min with moderateagitation in a 143 mM solution of succinic anhydride dissolved in1-methyl-2-pyrrolidinone, 20 mM sodium borate, pH 8.0. Slides wererinsed for 2 min in distilled water, immersed for 1 min in 95% ethanol,and dried with a stream of nitrogen gas.

The cDNA generated above was added to hybridization buffer containingCot-1 DNA (0.5 mg/ml final concentration), yeast tRNA (0.2 mg/ml), andpoly(dA)₄₀₋₆₀ (0.4 mg/ml). Hybridization was performed on a VentanaDiscovery system for 6 hr at 42° C. (Ventana Medical Systems, Tucson,Ariz.). Microarrays were washed to a final stringency of 0.1×SSC.Microarrays were scanned on a dual-channel, dynamic auto focus,fluorescent scanner at 10 um resolution (Agilent Technologies, PaloAlto, Calif.). Fluorescent intensity was determined using Imagene™software (BioDiscovery, Marina del Rey, Calif.).

Genechip Analysis in CSC Experiments

CSC-induced changes in gene expression were then determined in acomprehensive manner using hypervariable analysis, which is based on theobservation that gene expression for a majority of genes is relativelystable among replicates in untreated cells. Any measurable variation inthis large set of genes by micro array analysis reflects the combinedeffects of intrinsic normal biologic variation and extrinsictechnological variation in an unmanipulated cell. Genes that wereimpacted by exposure to CSCs, and whose mRNA expression varied over timein a statistically significant manner, which was greater than thisnormal biologic and technical variation, are termed “hypervariable”(HV).

Signals from independent samples can vary on a global-basis and,preferably, are adjusted to a common standard. Adjustment of expressionlevels in compared samples was performed as described. (See Dozmorov, etal. Bioinformatics 19:204-211, 2003, expressly incorporated by referencein its entirety). Briefly, compared samples were first normalized usinglow level noise signals (commonly referred to as additive noise (AN).The parameters of the AN were calculated from non-expressed genes whosesignal values exhibited a normal distribution. The mean and standarddeviation (SD) of the AN signals was obtained by nonlinear curve fittingafter exclusion of expressed genes from the distribution. Expressionvalues from a given chip were then normalized such that the ANdistribution had a mean of 0 and a SD of 1. Genes expressed 3 SD abovethe mean of AN are defined as expressed genes and used for furtheranalysis. A second scaling step was then performed on expressed genesthat were scaled to a common standard through a robust linear regressionanalysis.

Genes responsive to CSCs were also identified using an analysis oftemporally induced gene expression changes. This procedure utilized aninternal standard, denoted “the reference group” to define the levels oftechnologic and normal biologic variance in the experiment so that thesevalues can be used to define stimuli-induced variation in astatistically robust manner. The majority of genes in the control groupwere not sensitive to temporal changes. The reference group wastherefore composed of a group of genes that were statistically expressedsignificantly above the mean of AN in control samples, whose residualsapproximate a normal distribution based on the Kolmogorov-Smirnovcriterion, and that have low variability of expression over time asdetermined by an F-test. Variance in the reference group is due only totechnical variation and normal biologic variation and therefore thedistribution of expression of the reference group can be used toidentify genes that vary due to experimental conditions in a manner thatis statistically significantly higher than the technologic and normalbiologic variance of the system using an F-test. Genes identified usingthese procedures are denoted “hypervariable genes” or “HV-genes”.

F-means cluster analysis of HV-genes co-expression involved groupings ofgenes that varied in expression over time in a similar manner, based onthe technologic and normal biologic variation in the system, in a givencluster. The reference group defined above is once again used as areference to define statistically significant thresholds for clusteringparameters used in an F-test. In this manner, the variance of the systemis used to define the number of clusters thus removing the subjectivenature of most clustering methods. The method is not without somesubjective criterion as genes can belong to multiple clusters. In thismethod, a given gene is placed into the largest cluster such that thebroadest biologic phenomena of the system, that is those involving thelargest number of genes, can be distinguished. To do this, clustering isbegun by defining a simple parameter for each HV-gene. This parameter,denoted connectivity, is equal to the number of genes that vary inexpression in a similar manner as a given gene. Clusters are nucleatedstarting with genes of highest connectivity. Genes of lower connectivitywill be included in a given cluster if their expression varies over timein a manner similar to the gene used to nucleate the cluster, i.e. iftheir deviations of expression over time do not exceed the variation ofthe residuals in the reference group based on an F test.

F-clustering was used to identify the kinetic behavior of genes for eachstimulus. Correlation coefficient analysis was used to identify genesthat behave in a similar manner among groups. In this type of analysis,a Pearson correlation coefficient is used for clustering of genes withsimilar time-dependent behavior among groups. A correlation thresholdwas established using a Monte-Carlo simulation experiment such that thechances of identifying a false positive or false negative selection is<1. Matrices of correlation coefficients are calculated for theseclusters and are represented in a graphical output termed a connectivitymosaic such that patterns of correlated and non-correlated behavior ofgenes can be identified by visual inspection.

Discriminant function analysis (DFA) is a method that identifies asubset of genes whose expression values can be linearly combined in anequation, denoted a root, whose overall value is distinct for a givencharacterized group. DFA therefore, allows the genes that maximallydiscriminate among the distinct groups analyzed to be identified. (SeeMoore et al. Genet Epidemiol 23: 57-69, (2002), expressly incorporatedby reference in its entirety). In the experiments described herein, avariant of the classical DFA, named the Forward Stepwise Analysis, wasused for selection of the set of genes whose expression maximallydiscriminates among experimentally distinct groups. The Forward StepwiseAnalysis was built systematically. Specifically, at each step allvariables were reviewed to identify the one that most contributes to thediscrimination between groups. This variable was included in the model,and the process proceeds to the next step. The statistical significanceof discriminative power of each gene was also characterized by partialWilk's Lambda coefficients (see Cho et al., Optimal approach forclassification of acute leukemia subtypes based on gene expression data.Biotechnol Prog 18: 847-854, 2002), expressly incorporated by referencein its entirety, which are equivalent to the partial correlationcoefficients generated by multiple regression analyses. The Wilk'sLambda coefficient used a ratio of within group differences and the sumof within plus between group differences. Its value ranged from 1.0 (nodiscriminatory power) to 0.0 (perfect discriminatory power).

Of the 21,349 genes and open reading frames (ORFs) on the high-densityarray used in these experiments, a combined total of 4,894 (22.9%) wereclassified as HV after CSC treatment (see FIG. 34A). Individually, theexpression of 3,665 genes/ORFs was modulated by CSC-A contact (i.e.,17.2% of all the genes/ORFs on the array), and the expression of 3,668genes/ORFs was modulated by CSC-B contact (17.2%). These genes werehypervariable in at least one time point during the 12-hour exposureperiod to CSC-A and CSC-B respectively (see FIG. 36, Table 7). Theobservation that the expression of a large number of genes was alteredin a significant manner during the 12 h treatment demonstrated asignificant impact by CSCs on steady-state levels of mRNAs in NHBEcells. A majority of the HV genes (i.e., 2,439) were common to bothCSC-treated groups, providing evidence that the two CSCs affected cellslargely in a similar manner. However, unique non-overlapping sets of HVgenes were also identified after treatment with CSC-A (i.e., 1226 genes)and CSC-B (i.e., 1229 genes), which demonstrate that each tobaccoproduct has a specific quantitative and/or qualitative difference in thechemical constituents comprising the two CSCs and the cellular responsesthereto.

TABLE 7 Genes Common to CSC-A and CSC-B exposed cells, which areassociated with a tobacco-related disease GenBank Gene accession no.Abbreviation Gene description Disease NM_001610 ACTA2 Actin, alpha 2,smooth muscle, aorta Lung Cancer NM_005181 CA3 Carbonic anhydrase III,muscle specific Lung Cancer NM_005199 CHRNG Cholinergic receptor,nicotinic, gamma polypeptide Lung Cancer NM_002594 PCSK2 Proproteinconvertase subtilisin/kexin type 2 (PC2) Lung Cancer NM_004624 VIPR1Vasoactive intestinal peptide receptor 1 (VPAC1) Lung Cancer NM_004448ERBB2 V-erb-b2 erythroblastic leukemia viral oncogene Lung (HER2/NEU)homolog 2 Cancer NM_024083 ASPSCR1 Alveolar soft part sarcoma chromosomeregion, Lung candidate 1 Cancer NM_003872 NRP2 Neuropilin 2 Lung CancerU33749 TITF1 Thyroid transcription factor 1 Lung Cancer NM_002639SERPINB5 Serine (or cysteine) proteinase inhibitor, clade B Lung(ovalbumin), member 5, (maspin) Cancer AF135794 AKT3 V-akt murinethymoma viral oncogene homolog 3 Lung (protein kinase B, gamma) CancerNM_001618 ADPRT ADP-ribosyltransferase (NAD+; poly (ADP- Lung ribose)polymerase) PARP1 Cancer NM_016434 TNFRSF6B Tumor necrosis factorreceptor superfamily, Lung member 6b, decoy Cancer NM_003072 SMARCA4SWI/SNF related, matrix associated, actin Lung (BRG1) dependentregulator of chromatin, subfamily a, Cancer member 4 NM_004061 CDH12Cadherin 12, type 2 (N-cadherin 2) Lung Cancer U28749 HMGICHigh-mobility group (nonhistone chromosomal) Lung protein isoform I-CCancer NM_002592 PCNA Proliferating cell nuclear antigen Lung CancerNM_033215 PPP1R3F Protein phosphatase 1, regulatory (inhibitor) Lungsubunit 3F (PPP1R3F), mRNA Cancer NM_006218 PIK3CA Phosphoinositide3-kinase, catalytic, alpha Lung polypeptide Cancer NM_005506 CD36L2 CD36antigen (collagen type I receptor, Lung thrombospondin receptor)-like 2(lysosomal Cancer integral membrane NM_004994 MMP9 Matrixmetalloproteinase 9 Lung Cancer NM_003810 TNFSF10 Tumor necrosis factor(ligand) superfamily, Lung member 10 (TRAIL) Cancer NM_002961 S100A4S100 calcium binding protein A4 (calcium protein, Lung calvasculin,metastasin, murine placental homolog) Cancer NM_007084 SOX21 SRY (sexdetermining region Y)-box 21 Lung Cancer NM_003682 MADD MAP-kinaseactivating death domain (DENN) Lung Cancer BC002712 MYCN V-mycmyelocytomatosis viral related oncogene, Lung neuroblastoma derived(avian) Cancer NM_004353 SERPINH1 Serine (or cysteine) proteinaseinhibitor, clade H), Oral member 1, HSP47 Cancer NM_000640 IL13RA2Interleukin 13 receptor, alpha 2 Asthma NM_002046 GAPDGlyceraldehyde-3-phosphate dehydrogenase Asthma NM_021804 ACE2Angiotensin I converting enzyme (peptidyl- Coronary dipeptidase A) 2Heart Disease NM_017614 BHMT2 Betaine-homocysteine methyltransferase 2Coronary Heart Disease NM_020974 CEGP1 CEGP1 protein Coronary HeartDisease NM_018641 C4S0 Chondroitin 4-O-sulfotransferase 2 Coronary HeartDisease NM_006874 ELF2 E74-like factor 2 (ets domain transcriptionfactor), Coronary NERF Heart Disease*The sequences of the genes above are available from GenBank using thereferenced Gene ID No. and said sequences are hereby expresslyincorporated by reference in their entireties.

The 1229 genes that were induced by exposure to CSC-B but not CSC-A werealso analyzed with the commercially-available microarray data analysissoftware Genespring (version 7.2, Agilent Technologies), whichidentifies genes that are associated with a tobacco-related disease. Ofthe 1229 unique genes that were induced by exposure to CSC-B but notCSC-A, a total of 33 genes were identified as being associated withcancer (see Table 8).

TABLE 8 Genes modulated by contact with CSC-B but not CSC-A, which areassociated with a tobacco-related disease GeneBank # Name DescriptionNM_00359 CUL4A Cullin 4A NM_00405 CDR1 Cerebellar degeneration-relatedprotein (34 kD) NM_00521 CSF1R Colony stimulating factor 1 receptor,formerly McDonough feline sarcoma viral (v-fms) oncogene homol NM_00626TFDP2 Transcription factor Dp-2 (E2F dimerization partner 2) NM_01225SNW1 SKI-interacting protein NM_00482 NTN1 Netrin 1 NM_00284 RAP1ARAP1A, member of RAS oncogene family AF308602 NOTCH1 Notch homolog 1,translocation- associated (Drosophila) NM_01438 LAMP3Lysosomal-associated membrane protein 3 NM_00371 PPAP2A Phosphatidicacid phosphatase type 2A NM_00164 ARHA Ras homolog gene family, member ANM_01633 LOC51191 Cyclin-E binding protein 1 NM_01865 ERBB2IP Erbb2interacting protein NM_01242 SETDB1 SET domain, bifurcated 1 AF156165DCTN4 Dynactin 4 (p62) NM_00205 FOXO1A Forkhead box O1A(rhabdomyosarcoma) AF163473 PPP2R1B Protein phosphatase 2 (formerly 2A),regulatory subunit A (PR 65), beta isoform NM_03328 PML Promyelocyticleukemia AK024486 GLTSCR2 Glioma tumor suppressor candidate region gene2 NM_00343 ZNF151 Zinc finger protein 151 (pHZ-67) U18018 ETV4 Etsvariant gene 4 (E1A enhancer binding protein, E1AF) NM_00523 EWSR1 Ewingsarcoma breakpoint region 1 BC013971 HOXA10 Homeo box A10 AJ420488EEF1A1 Eukaryotic translation elongation factor 1 alpha 1 NM_00548 ST5Suppression of tumorigenicity 5 NM_00578 HNRPA3 Heterogeneous nuclearribonucleoprotein A3 NM_00094 RARA Retinoic acid receptor, alphaNM_00675 N33 Putative prostate cancer tumor suppressor NM_00228 JUNV-jun sarcoma virus 17 oncogene homolog (avian) AL110274 ALDH1A2Aldehyde dehydrogenase 1 family, member A2 NM_01428 RBX1 Ring-box 1NM_01787 FLJ20429 Hypothetical protein FLJ20429 NM_00437 BCR Breakpointcluster region *The sequences of the genes above are available fromGenBank using the referenced Gene ID No. and these sequences are herebyexpressly incorporated by reference in their entireties.

Notably, it was discovered that CSC-B induced expression of theproto/oncogenes Cullin 4A, C-jun, Hoxa10, and PPP2R1B, whereas CSC-A didnot. Cullin 4A has been described in non-small cell lung cancer (seeSinghal et al., Cancer Biol. Ther. 2(3):291-298 (2003)); C-jun has beenfound to be amplified or over expressed in small cell lung cancer (seeCook et al., Curr. Probl. Cancer 17(2):69-141 (1993)); Hoxa10 has beenfound to be amplified or over expressed in leukemia (see Calvo et al.,Proc. Natl. Acad. Sci. USA 97(23):12776-12781 (2000)); and alteredexpression of PPP2R1B is involved in lung and colorectal carcinomas (seeCalin et al., Oncogene 19(9):1191-1195 (2000); all of these referencesare expressly incorporated by reference in their entireties).Accordingly, these results demonstrate that the tobacco productcomprising chemically modified tobacco (Brand A cigarette), which wasused to generate CSC-A, has a reduced potential to contribute to atobacco-related disease as compared to the tobacco product (Brand Bcigarette) used to generate CSC-B because CSC-A induces expression offewer genes associated with a tobacco-related disease (e.g., 33 fewergenes associated with cancer). Notably, the tobacco product used togenerate CSC-A (Brand A) does not induce key genes that have beenassociated with cancer in humans (e.g., the proto/oncogenes Cullin 4A,C-jun, Hoxa10, and PPP2R1B); whereas the tobacco product used togenerate CSC-B (Brand B) induces expression of these proto/oncogenes.Further, these results demonstrate that the methods described herein canbe used to effectively identify a tobacco product that is less likely ormore likely to contribute to a tobacco related disease (e.g., cancer).That is, this example demonstrates that the approaches described hereincan be used to identify a reduced risk tobacco product, which can be atobacco product that is less likely to contribute to a tobacco-relateddisease because it modulates fewer genes associated with atobacco-related disease (e.g., cancer) or induces fewer modifications toa gene product, which are associated with a tobacco-related disease, ascompared to a second tobacco product. To confirm these data, moreexperiments were conducted on the tobacco product used to generate CSC-A(Brand A) to determine whether it was in fact less likely to contributeto a tobacco-related disease (e.g., cancer), as compared to the tobaccoproduct used to generate CSC-B (Brand B). These experiments arediscussed in the following example.

Example 6

This example describes experiments that were conducted on mice todemonstrate that the tobacco product used to generate CSC-A (Brand A) isa reduced risk tobacco product in that it was less likely to contributeto a tobacco-related disease, as compared to a conventional tobaccoproduct of the same class (e.g., “full flavor” cigarette), Brand B,which was used to generate CSC-B in the previous examples. In summary,the response of previously initiated SENCAR mice to repeated topicalapplications of Brand-A or Brand-B Cigarette Smoke Condensates (CSC-A orCSC-B), was tested over a period of 24 consecutive weeks. One week aftera single initiating dose of 50 μg 7,12-dimethylbenzanthracene(7,12-DMBA), female SENCAR mice were exposed to the followingthree-times-per-week treatment regimen: Negative-Initiation Control (0.1ml acetone promotion); Positive Control (1 μg TPA promotion); Test(Brand-A CSC promotion, low-dose [10 mg] and high-dose [20 mg]); or Test(Brand-B CSC promotion, low-dose [10 mg] and high-dose [20 mg]). Thecondensates and positive control articles were dissolved in acetone andapplied three times a week to the shaved dorsal skin of female SENCARmice. In addition, a vehicle control group was initiated and promotedwith acetone only. The effects of treatment with the various articles onsurvival and group mean body weights did not appear to be significantlyaffected by the Test CSC's during the duration of the study phase.

The extent of tumor promotion by the cigarette smoke condensates wasquantitated by the incidence of tumor-bearing animals per group, themultiplicity of tumors per animal, and the latency period until theappearance of tumors. All quantitative scoring was based on gross tumordetection, gross tumor numbers, and gross characterization of tumorswhich was shown to be accurate by histopathologic examination. Theresponse to the Test CSCs was evident in 13-87% incidence ofDMBA-initiated animals exhibiting actual tumors in the effective animalsof those groups after 25 weeks compared to a 3% incidence (a singleanimal) exhibiting actual tumors in the Negative Control(DMBA-Initiated) group. There were no incidences of animals exhibitingactual tumors in the acetone-initiated group.

The SENCAR mouse is an acceptable short-term in vivo model forevaluating the promoting potential of a cigarette on multi-stageepidermal carcinogenesis. This assay system takes advantage of a mousestrain that is extremely sensitive to the two-stage induction of skintumors. SENCAR mice were bred for increased sensitivity to skin tumorinitiation and promotion. The strain originated from Rocklandall-purpose mice which were inbred for sensitivity to skin tumorinitiation by DMBA and promotion by12-0-tetradecanoyl-phorbol-13-acetate (TPA) in 1959. In 1971, thesesusceptible mice were outbred with Charles River CD-1 mice to producehybrid vigor. These mice have been bred for use in skin carcinogenesisstudies of up to 12 months duration.

Accordingly, the SENCAR mouse skin painting bioassay was utilized todetermine the relative promoting potential of various cigarette smokecondensate (CSC) preparations applied topically for 24 consecutiveweeks. The mice in Groups, as described below, were initiated with asingle application of 50 μg 7,12-dimethylbenzanthracene (DMBA). One weekafter initiation, the animals of each group received three topicalapplications per week of either acetone (Negative Controls), TPA(Positive Control), or one of two dose levels of cigarette smokecondensates (CSC) from the Test cigarettes. The mice in Group 1 wereinitiated with acetone vehicle rather than DMBA and received acetonepromotion thereafter.

Late in the quarantine period, the animals were weighed and randomlydistributed into nine study groups using a computerized randomizationprogram. This program insured that no statistically significantdifferences in the group mean body weights existed between the studygroups at study start. Animals with body weights that were ±20% of themean body weight of the animal pool were assigned to the study.Following assignment to a group (as listed in TABLE 9), each animal wasidentified by a uniquely numbered tail tattoo. A color-coded card whichlisted the study number, animal number, group designation and treatmentwas displayed on each cage.

TABLE 9 Test Group Animal No. of Article No. No. Test Group Animals No.1  1-30 Negative-Vehicle Control, 30 Not Acetone Initiation and Appli-Acetone Promotion (0.1 ml cable each) 2 31-60 Negative-InitiationControl, 30 Not DMBA Initiation (50 μg) Appli- Acetone Promotion (0.1ml) cable 3 61-80 Positive Control, DMBA 20 Not Initiation (50 μg)Appli- TPA promotion (1 μg) cable 4  81-120 Low Dose Brand A, DMBA 40AA49LY Initiation (50 μg) Brand A CSC Promotion (10 mg) 5 121-160 HighDose Brand A, DMBA 40 AA49LY Initiation (50 μg) Brand A CSC Promotion(20 mg) 8 241-280 Low Dose Brand B, DMBA 40 AA52CE Initiation (50 μg)Brand B CSC Promotion (10 mg) 9 281-320 High Dose Brand B, DMBA 40AA52CE Initiation (50 μg) Brand B CSC Promotion (20 mg)

The Test cigarette smoke condensates at 100 and 200 mg total tarcontent/ml were collected and prepared by Arista Laboratories at afrequency of approximately every 8 weeks. Upon receipt, the CSC sampleswere stored at ≤−20° C. until further sub-aliquoted by BioReliance (5.0ml per vial for both the low and high doses) and stored at ≤−20° C. Thedose preparations, as received from Arista Laboratories, were dividedinto 26 tightly sealed amber vials, with an expiration date ofapproximately 13 weeks and stored at ≤−20° C. This allowed the use ofone vial per dosing day and two backups which could be used in case ofspillage. All dosing solutions were used within eight weeks ofpreparation. The Positive Control article (TPA) was diluted with acetoneto produce the desired concentration of 10.0 μg/ml once (prior toinitiation of dosing) and delivered to the animal laboratory and storedat room temperature (an extra vial was stored at ≤−20° C.).

The mice from Groups 2-9 received a single topical application of DMBA(50 μg/0.1 ml acetone/animal) as an initiator on Day 1 of the study. Themice from Group 1 received a single topical application of acetonevehicle (0.1 ml) as an initiator. After one week, the animals were dosedtopically three times a week (Monday, Wednesday and Friday except forHolidays) for 24 consecutive weeks with the appropriate Vehicle Control,Positive Control or Test article.

The dorsal application site (approximately 2×3 cm) was shaved 3 daysprior to the single application of the initiator, and at least once aweek thereafter, at least one day prior to application of theappropriate dosing solution or vehicle. Shaving was performed on allanimals with an Oster Model 76059 small animal electric clippers (OsterCo., Racine, Wis.) using a narrow blade.

The animals were weighed at study initiation and at weekly intervals forthe next 11 weeks (12 total data collection points), and once every fourweeks thereafter and at terminal sacrifice. The animals were observedtwice daily (including weekends and holidays) for mortality andmoribundity, once in the morning before 10:00 a.m. and once in theafternoon after 2:00 p.m. (at least six hours apart). Clinicalobservations performed cage-side to detect abnormalities other than skintumor responses were made once daily for the first 5 weeks of the study(Days 1-35) and hands on once every two weeks thereafter (beginning onDay 36). Clinical signs noted at times other than the scheduledobservation timepoints were recorded on the Unscheduled ObservationsSheet.

On Day 1 and at weekly intervals thereafter, the mice were examinedgrossly for the presence of skin tumors. Pertinent information such asdate of observation, lesion location, morphology, and type were recordedfor each lesion at each observation time. At necropsy, allrepresentative skin from the application site, skin from an untreatedarea, and other lesions taken for histopathologic evaluation wereindicated on the necropsy data sheet. Lesions were identified in amanner which allowed correlation of the individual lesion-specifichistopathologic findings with data collected during the in-life phase ofthe study.

A tissue mass (in vivo) was considered to be a tumor (papilloma) whenthat mass attained a 2 mm diameter and protruded from the surface of theskin. The date at which a 2 mm diameter was attained was recorded andrepresented the end of the “tumor latency period” for that animal andthe tumor was scored as a latent papilloma. If a latent tumor remainedcountable for three (3) consecutive weeks, it was considered an actualtumor. Such a tumor remained in the total count of actual tumors forthat animal even if it subsequently decreased in size, disappeared, orthe animal died or was sacrificed early. The record of skin lesion dataserved to differentiate papillomas from carcinomas and latent tumorsfrom actual tumors. In vivo differentiation of papillomas and carcinomaswas made on the basis of palpation, evidence of subcutaneous invasion,and ulceration.

Group 3 (the positive control) served as a qualitative indicator of thetest system's response to a known and chemically defined initiator(DMBA) and promotor (TPA). Considering the time course and magnitude ofthe response in SENCAR mice, treated as described above, collection ofskin lesion data in the positive control was discontinued after 90-100%of the animals in the group exhibited tumors and the mean number oftumors per animal was at least 8. Since this group was not countedthrough the entire study, it was not included in any group comparisonsnoted below.

The number and location of skin papillomas (benign tissue masses havingattained a diameter >2 mm and protruding from the surface) andcarcinomas (malignant tissue masses with gross evidence of invasivegrowth and tissue necrosis due to growth outstripping vascular supply)were documented weekly. The reliability of gross diagnoses of tumors wasconfirmed by representative histopathologic examination of individuallyidentified and historically tracked skin lesions. Tumor data forspecific groups were calculated based on the appearance of tumors ofeither type. The following parameters were recorded or calculated forall groups (with the exception of Group 3, Positive Control):

-   -   1. Date of tumor appearance for all tumors on all mice.    -   2. Date of appearance of latent and actual tumors.    -   3. Date of death or sacrifice for each mouse.    -   4. Time interval from Day 1 of the study until the date of the        appearance of; (1) latent papillomas and carcinomas and, (2)        actual papillomas or carcinomas on each mouse.    -   5. Latency for all latent or actual tumors (i.e., this was        defined as the time from Day 1 to the time a mass qualified as a        latent tumor and subsequently as an actual tumor). Three methods        for numerically scoring latency were used:        -   a. The time elapsed until the appearance of the first tumor            of a specific type in a group.        -   b. The mean time elapsed until the appearance of all first            tumors of a specific type from all animals in a group            developing one or more such tumors.        -   c. Time elapsed to attain 50% of the maximum incidence of            animals in a group with one or more tumors of a specific            type.    -   6. Percent of mice developing one or more latent and/or actual        tumors (Incidence) equals:        -   Number of mice with at least one latent and/or actual            tumor×100        -   Number of mice surviving at the time the first non-positive            control group shows a tumor    -   7. Tumors per tumor-bearing animal=Number of total or        specific-type tumors Number of animals bearing that type of        tumor

Group means and standard deviations were calculated for body weights andskin tumor data. A Fisher's Exact test was performed to analyze thepercent of surviving animals in each group which developed latent and/oractual tumors and percent of animals started on study which developedactual tumors. Analysis of Variance tests (ANOVA) were performed inorder to determine if differences in group means existed for theselected parameters. If a significant F ratio was obtained (p<0.05), aDunnett's t-test was used for pair-wise comparisons of treatment testCSC groups to the Negative Control (non-Initiated DMBA) and test CSCgroups with each other.

Incidence of Tumor-Bearing Animals

Statistical analysis of the incidence of animals bearing actual tumors(Fisher's Exact Test, p<0.05) indicated a significant increase in boththe low- and high-dose groups receiving CSC-B when compared to thenegative vehicle control group. Of the groups receiving the CSC-A, onlythe high-dose exhibited a significantly increased number of animalsbearing actual tumors when compared to the negative vehicle controlgroup. When comparing the incidence of animals bearing actual tumors inthe low-dose CSC treatment groups to each other, a significant increasewas noted in the groups that received CSC-B when compared to the groupthat received CSC-A. The same results were obtained when making the samecomparisons in the groups receiving the high-dose CSC treatment. Thesefindings are presented in TABLE 10.

TABLE 10 Statistical Results of Analysis of Percent of Animals BearingActual Tumors Percent of Animals Bearing Group Treatment ActualTumors^(a, b) 1 Negative Vehicle Control  0% 4 Low-Dose Brand A 13% 5High-Dose Brand A 40% 8 Low-Dose Brand B 53% 9 High-Dose Brand B 78%^(a)Represents the percent of animals started on study that developed atleast one actual tumor. ^(b)Significantly increased when compared to thegroup indicated in the superscript (Fisher's exact test, p < 0.05).

Statistical analysis of the incidence of animals bearing actual and/orlatent tumors (Fisher's Exact Test, p<0.05) comparing the CSC treatmentgroups to the negative vehicle control indicated the same results as theanalyses of animals bearing actual tumors discussed above.

Tumor Multiplicity

Statistical analysis of the number of actual tumors (papillomas andcarcinomas combined) per animal, after 24 weeks, revealed significantincreases (ANOVA, p≤0.05) in the groups treated with the high-dose CSC-Bwhen compared to the negative control group (acetone-initiated Group 1).The number of actual tumors per animal in the group treated with thehigh-dose CSC-A group was statistically comparable to the negativevehicle control group. Analysis of the number of actual tumors peranimal in the low-dose CSC treatment groups indicated the group treatedwith the low-dose Brand B CSC exhibited a statistically significantlyincreased number of actual tumors when compared to the negative controlgroup. Group means, standard deviations, and statistical results arepresented in TABLE 11.

TABLE 11 Statistical Results of Analysis Number of Actual Tumors perAnimal Mean Number of Actual Group Treatment Tumors per Animal ^(a) 1Negative Vehicle Control 0.00 ± 0.00 4 Low-Dose Brand A 1.03 ± 3.90 5High-Dose Brand A 2.58 ± 8.05 8 Low-Dose Brand B 3.80 ± 7.22 9 High-DoseBrand B 7.46 ± 7.86 ^(a) Significantly increased when compared to thegroup indicated in the superscript.

When comparing the high-dose CSC treatment groups against each other, astatistically significantly increased number of actual tumors per animalwas noted in the high-dose groups treated with the Brand B CSC whencompared to the high-dose Brand A group. No statistically significantdifferences in the numbers of actual tumors were noted in the low doseCSC treatment groups when compared to each other. Statistical analysisof the number of actual and/or latent tumors (ANOVA, p≤0.05) indicatedthe same results as the analyses of the number of actual tumors peranimal, as discussed above. Results are presented in the followingTable.

TABLE 12 Statistical Results of Analysis Number of Latent and ActualTumors per Animal Mean Number of Actual Group Treatment Tumors perAnimal ^(a) 1 Negative Vehicle Control 0.00 ± 0.00 4 Low-Dose Brand A1.20 ± 4.33 5 High-Dose Brand A 2.75 ± 8.13 8 Low-Dose Brand B   4.73 ±8.35 ¹ 9 High-Dose Brand B   8.49 ± 8.70 ^(1, 5) ^(a) Significantlyincreased when compared to the group indicated in the superscript.

Latency Period Until Appearance of Tumors

Mean latency per group when defined as the time elapsed until theappearance of the first actual tumor per animal was 18 weeks in thelow-dose of both CSC-A and CSC-B treatment groups. In the high-dose CSCtreatment groups, mean actual tumor latency was 19 and 15 weeks in thegroups treated with CSCs obtained from Brands A and B, respectively.

Thus, the promotional capacity of the Brand A CSC was statisticallycomparable to the negative vehicle control group in terms of theincidences of tumor-bearing animals (at the low-dose level) and thenumber of tumors per animal (both dose levels). Statistical analysiscomparing the groups that received the CSCs to each other revealedsignificant increases in the high-dose Brand B group when compared tothe high-dose Brand A group in terms of percent of animals bearingdesignated tumor types and the number of those tumors per animal. Also,at the high-dose level, the Brand A CSC mean latency period (until theappearance of the first tumor per animal) was longer than the latencyperiod of the Brand B CSC treatment groups. The data provided in thisexample confirm that the in vitro methods described herein, whichutilize cell cultures that are contacted with CS, CSC, TS, TSC or TPM(see Examples 4, 5, and 8-13), accurately identify a tobacco productthat has less potential to contribute to a tobacco-related disease thananother tobacco product. The data provided in this example also confirmthat the in vitro methods described herein (see Examples 4, 5, and8-13), can be used to develop tobacco products that have a reducedpotential to contribute to a tobacco-related disease and provide furtherevidence, in particular, that Brand A is a reduced risk tobacco product,as compared to Brand B.

Subsequent to exposure in vivo, the human body attempts to detoxify,neutralize, and eliminate cigarette smoke toxins through the action ofPhase I and Phase II enzymes functioning in various metabolic pathways.During this detoxification process, however, a number ofpro-carcinogenic compounds in tobacco smoke are bioactivated intoreactive electrophiles that have potent carcinogenic potential inexposed cells. Thus, in order to dissect the full biological potentialof complex chemical mixtures, such as a cigarette smoke condensate, itis desirable to evaluate the pattern of gene expression after tobaccosmoke condensate exposure in an environment that contains a mixture ofenzymes that mimic the detoxification process in mammalian cells. The S9microsomal fraction from Aroclor 1254-treated rats, provides a set ofenzymes that mimic the detoxification process in mammalian cells.Accordingly, experiments were conducted in the presence of the S9microsomal fraction, as described in the following example, to elucidatehow the genetic fingerprint of particular tobacco products shift in thepresence of a mixture of enzymes that mimic the detoxification processin mammals.

Example 7 S9 Microsomal Fraction Experiments

NHBE cells were exposed to cigarette smoke condensate (CSC) inconjunction with an S9 microsomal fraction so as to identify the effectdetoxification enzymes have on the pattern or level of gene expression.As a control to discriminate the effects of the S9 microsomal fractionon gene expression, alone, some experiments were conducted on NHBE cellsin the presence of the S9 microsomal fraction in the absence of contactwith a tobacco condensate. As described above, an HV analysis wasperformed on microarray results obtained from cells treated only withthe S9 microsomal fraction for 2, 4, 8, and 12 hours.

Several interesting observations emerged from this analysis. First, theexpression of 1680 (7.9%) genes became HV sometime during the 12-hourexposure period with the S9 microsomal fraction (see FIG. 34B). Second,FIG. 34B also shows that 1297 of these 1680 genes were also HV in one orboth CSC treatments, which is not surprising since all three treatmentconditions (i.e., CSC-A, CSC-B, and S9) had the same concentration of S9microsomal fraction. Third, even though the CSCs and the S9 microsomalfraction induce a HV state in a large common set of genes, CSCs and theS9 microsomal fraction did not affect these genes in similar waysindicating differential kinetic effects between the S9 microsomalfraction alone and the S9 microsomal fraction in conjunction with CSCs.

Subsequent to determining that the complex mixture of toxins andcarcinogens in CSCs had a broad impact on the transcriptome of NHBEcells, it was contemplated that a sustained treatment to CSCs (e.g.,over a 12-hour period) would also allow detection, not only ofalterations such as induction and suppression, but of geneinduction/suppression with transient, sustained, or periodiccharacteristics. Accordingly, the kinetic effects of gene expressionprofiles generated from cells treated with CSC-A, CSC-B, or S9microsomal fraction from 0-12 hours using F-cluster analysis weredefined, which is a statistically robust method for defining clusters ofgenes with similar expression patterns over time. These experiments aredescribed in the following example.

Example 8 Gene Expression Kinetics in CSC-Treated Cells

In this analysis, the normal variance of the system was calculated andused to identify a statistical threshold for cluster selection at whichgroups of genes were likely to cluster by chance. This threshold wasthen used for further analysis to ensure the statistical robustness ofthe clustering process. The biologic significance of the cluster isrelated to cluster size, as the largest clusters identified representsynchronous changes in the greatest number of cellular processes. (SeeSpellman et al., Mol Biol Cell 9: 3273-3297, 1998). Specifically, largerclusters represent, in a statistically robust manner, the mostsignificant experimentally induced processes in these cells. WhenF-cluster analysis was applied to the total HV set of 4894 genes/ORFs,306 clusters were defined by statistical analysis, the majority of whichcontained less than 50 member genes. Cluster numbers were arbitrarilyassigned from −150 to 150, with the corresponding positive and negativenumbers representing complementary gene expression patterns (e.g.,steady increase in expression over time compared to a steady decrease inexpression).

In each of the three treatment conditions, clusters containing 50 ormore genes were chosen for further characterization because this cutoffgenerated a sufficient number of large clusters that adequatelyrepresented the major kinetic changes caused by each treatment (seeFIGS. 2 A-C and TABLE 13). As predicted, gene expression changes inducedby CSCs were complex, with the majority of clusters in CSC-treated cellsbeing multi-modal (see FIGS. 2A and B). For example, in CSC-A-treatedcells, genes in clusters 1, 3, 7, 12, 15, and 22 were up-regulatedwithin the first two hours, began to return to baseline, then were onceagain induced late in the experiment, indicating that initial treatmenteffected gene expression and some secondary effect (e.g., a CSCmetabolite or the action of early gene expression changes, reinitiated acellular response). (See FIG. 35A). While genes within each of theseclusters showed early increases in expression (within the first 2 h oftreatment), indicating that CSC-A treatment had immediate effects oncells, Clusters 18, 30, 35, and 39 showed a later increase in geneexpression (i.e., ≥4 h). FIG. 35B shows that in CSC-B treated cells,cluster analysis revealed that gene expression peaks primarily between4-8 hours, as opposed to a 2 hour peak in CSC-A treated cells, providingevidence that some of the effects of CSC-B treatment were delayed withrespect to those of CSC-A (e.g., see clusters 4, 5, 9, 10, 16, and 32).These data are in distinct contrast to the major clusters of genes inS9-only treated cells, which displayed simple kinetics, i.e., expressiondecreasing or increasing continuously over time (see FIG. 35C). Although66% of HV genes affected by CSC-A and CSC-B were identical (see FIG.34), it is clear from FIG. 35 that the expression kinetics for thesegenes were nevertheless distinct for the two different CSCs. This isevidenced by the fact that the predominant coordinated behavior inCSC-A-treated cells is represented by the largest cluster (i.e., cluster1), that contains 1063 HV genes and whose expression peaked at 2 hourspost-treatment. This is in contrast to CSC-B-treated cells in which casethe predominant behavior of genes is represented by cluster 2, whichcontains 1,036 genes and whose expression peaked at 4-8 hours,indicating that some of the effects of CSC-B treatment are delayed withrespect to those of CSC-A.

TABLE 13 HV Genes Specific for CSC-A and CSC-B Treatment GenBank Geneaccession no. abbreviation Gene description AB032985 NXPH3 Neurexophilin3 AB046848 KIAA1628 KIAA1628 protein AB058772 SEMA6C Sema domain,transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6CAF178532 BACE2 Beta-site APP-cleaving enzyme 2 BC015737 Homo sapiens,ninjurin 2, clone MGC: 22993 IMAGE: 4907813 BC015929 NR1D2 Nuclearreceptor subfamily 1, group D, member 2 BC017732 STRBP Spermatidperinuclear RNA binding protein M23326 TRDV3 T cell receptor deltavariable 3 NM_000341 SLC3A1 Solute carrier family 3 (cystine, dibasicand neutral amino acid transporters, activator of cystine), member 1NM_000663 ABAT 4-aminobutyrate aminotransferase NM_000922 PDE3BPhosphodiesterase 3B, cGMP-inhibited NM_000981 RPL19 Ribosomal proteinL19 NM_001383 DPH2L1 Diptheria toxin resistance protein required fordiphthamide biosynthesis-like 1 (S. cerevisiae) NM_002046 GAPDGlyceraldehyde-3-phosphate dehydrogenase NM_002757 MAP2K5Mitogen-activated protein kinase kinase 5 NM_002890 RASA1 RAS p21protein activator (GTPase activating protein) 1 NM_003286 TOP1Topoisomerase (DNA) I NM_003408 ZFP37 Zinc finger protein 37 homolog(mouse) NM_004057 CALB3 Calbindin 3, (vitamin D-dependent calciumbinding protein) NM_004066 CETN1 Centrin, EF-hand protein, 1 NM_004083DDIT3 DNA-damage-inducible transcript 3 NM_004282 BAG2 BCL2-associatedathanogene 2 NM_004846 EIF4EL3 Eukaryotic translation initiation factor4E- like 3 NM_004939 DDX1 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 1NM_005476 GNE UDP-N-acetylglucosamine-2-epimerase/N- acetylmannosaminekinase NM_005619 RTN2 Reticulon 2 NM_007217 PDCD10 Programmed cell death10 NM_007275 FUS1 Lung cancer candidate NM_012192 FXC1 Fracture callus 1homolog (rat) NM_012288 KIAA0057 TRAM-like protein NM_013366 APC2Anaphase-promoting complex subunit 2 NM_013401 RAB3IL1 RAB3A interactingprotein (rabin3)-like 1 NM_014395 DAPP1 Dual adaptor of phosphotyrosineand 3- phosphoinositides NM_015057 KIAA0916 KIAA0916 protein NM_017491WDR1 WD repeat domain 1 NM_017581 CHRNA9 Cholinergic receptor,nicotinic, alpha polypeptide 9 NM_020122 PCMF Potassium channelmodulatory factor NM_020685 HT021 HT021 NM_021120 DLG3 Discs, large(Drosophila) homolog 3 (neuroendocrine-dlg) NM_031310 PLVAP Plasmalemmavesicle associated protein

Accordingly, these experiments demonstrated that not only do differenttobacco products induce different genes, gene expression patterns, andkinetics of gene expression but different tobacco products have adifferent impact on a cell or a tobacco consumer. That is, theprocedures described above can be used to obtain a genetic signature,pattern, or profile for a plurality of tobacco products and, becausesome of the modulated genes are associated with the induction orrepression of a tobacco-related disease, this data can be comparedand/or analyzed to identify a tobacco product with a reduced potentialto contribute to a tobacco-related disease.

Since clusters with a large number of member genes reflect predominantbiological behavior patterns that are likely to be functionallyinterrelated, it was contemplated that the cluster 1 set of 1063 genesfrom CSC-A-treated cells and the cluster 2 set of 1036 genes fromCSC-B-treated cells corresponded to important biological phenomenacommon to the two CSCs. If this were correct, then despite the fact thatCSC-A and CSC-B treatments modulate genes in a temporally distinctmanner, the two clusters should contain many of the same genes. Todemonstrate this point, the experiments in the following example wereconducted.

Example 9 Analysis of Cluster 1 and Cluster 2 in CSC-Treated Cells

Upon analysis of cluster 1 and cluster 2 in CSC-treated cells, it wasfound that a set of 554 genes (approximately 50% of the genes in eachcluster) were present in both cluster 1 (from CSC-A) and cluster 2 (fromCSC-B). A total of 330 genes from this set of 554 genes (59.5%) haveknown functions while the remaining 224 are ORFs.

Functional classification of these 330 genes common to cluster 1 andcluster 2 indicates that 10% have functional roles in proliferation,12.4% in transcription, 4.5% in apoptosis, and 5.1% in damage/repairresponses. In addition, as shown in TABLE 7, 34 (10%) of the identifiedgenes are documented as having roles in diseases that are associatedwith/long-term tobacco exposure (e.g., lung cancer, coronary heartdisease, and asthma).

In clear contrast to both CSC-A and CSC-B treated cells, the S9microsomal fraction-treated cells show a pronounced tendency towardssuppression of gene expression. An F-clustering analysis of the S9microsomal fraction data (shown in FIG. 35C) resulted in only fourclusters that contained 50 or more genes. Clusters 2, 5, and 44 all showdecreases in gene expression level with a nadir at 4-8 h. Cluster 18contains genes that show an increase in gene expression levels, butwhose expression peaks at 12 h, which is notably different from therobust early gene responses elicited by treatment with both CSCs.Additional evidence that the overall effects of S9 microsomal fractionand CSC exposure on gene expression levels are quite distinct wasobtained when traditional hierarchical clustering algorithms were usedto compare the overall differences in HV gene expression in eachtreatment group over the entire 12-hour time course. FIG. 36 shows theresults of this analysis for the common subset of genes that were HV inall three treatment groups (i.e., the 873 genes denoted in FIG. 34).Notably, the expression data for these 873 genes partition into twoseparate groups with S9-treated cells being clearly distinguishable fromCSC-A and CSC-B treated cells, which are similar to each other. The datafurther indicate that the S9 microsomal fraction exerts a largelysuppressive effect on the transcriptome of NHBE cells in contrast to apredominant inductive effect of CSC-A and CSC-B.

As discussed above, tobacco smoke condensates induce a range oftemporally distinct alterations to the homeostatic transcriptome of theNHBE cells, which were unique in that they were qualitatively andquantitatively dissimilar from the effects of exposure to a S9microsomal fraction. In an attempt to define a biological context forthese data, correlation analyses was used to identify genes whoseexpression changes were highly correlated in CSC-A and CSC-B treatedcells but not in S9-treated cells. This was achieved using a Monte Carloanalysis to establish a statistical threshold above which correlatedbehavior was unlikely to have occurred by chance. By this approach, geneexpression levels were randomized maintaining the same mean and standarddeviation. A correlation coefficient was then identified above which nogenes were correlated in the randomized data sets. The probability thatgenes that correlate in experimental data sets above this thresholdwould occur by chance is <1/total number of genes analyzed. Thefollowing example describes these experiments in greater detail.

Example 10 Defining CSC-Specific Toxicological Effects

The evidence provided in FIGS. 2 and 3 indicated that the effect ofexposure to CSC was significantly different than exposure to an S9microsomal fraction. Using the Monte Carlo analysis, as shown in TABLE13, forty HV genes were identified as having a modulation of geneexpression that was correlated in CSC-A and CSC-B treated cells but notin S9-treated cells. The similarities between the two tobacco-treatedsample groups can be visualized by applying a correlation coefficientanalysis to the genes within a given treatment, representing thisvisually in a correlation mosaic, and comparing the visual pattern ofthe mosaic to other such mosaics generated using data from differenttreatments. The correlation coefficients of these genes were presentedin a correlation mosaic map (see FIG. 37) in which genes with a highlycorrelated behavior were denoted by a grey pixel, and genes with highlynegatively correlated behavior by a black pixel. This mosaic provided away to assess the similarities of expression behavior of the correlatedgenes in CSC-A, CSC-B, and S9-treated cells by visual inspection.

The highly correlated expression characteristics of the CSC-impactedgenes identified by this analysis indicated that these genes were likelyto participate in pathways relevant to the effects specific to CSCexposure and not to exposure to the S9 microsomal fraction. Thesepathways were more clearly defined using PathwayAssist™ software(Stratagene, La Jolla, Calif.), a commercially available visualizationengine that scans and assesses documented literature and availablestandardized databases in order to filter, classify, and prioritizeproteins in terms of their functional relationships to known biologicalpathways. The results, provided in FIG. 38, highlight the fact that thisset of genes encodes proteins that play key roles in pathways that arerelevant to the documented pathological effects of cigarette smoke. Forexample, several of the genes listed in TABLE 13 are implicated in lungoncogenesis (e.g., FUS1, GAPD, & semaphorin), in various types ofdysfunctions in lung cells involving apoptosis (e.g., PDE3B, PDCD10), incell cycle control (e.g., MAP2K5, RASA1, APC2, RASA1), in DNA topologyand DNA repair (e.g., TOP1, DDIT3), and in cellular stress (e.g., BAG2).In addition, several genes are involved in neurosignaling (e.g.,neurexophilin, KIAA1628), neuroregeneration (e.g., semaphorin),neuropathology (e.g., BACE2, ABAT, DLG3), and inflammation (e.g., NINJ2,TRDV3, SLC3A1).

The induction of a range of neuroendocrine-related genes is interestingin light of the fact that many small cell lung cancers and somenon-small cell lung cancers exhibit a variety of pathological andmolecular features of pulmonary endocrine cells, and can be stimulatedby an autocrine/paracrine array of neuroendocrine peptides. Accordingly,expression of neuroendocrine markers has been shown to be useful in thedifferential diagnosis of lung cancers. The gene set shown in TABLE 13also includes CHRNA9, a human nicotinic acetylcholine receptor expressedin several tissues including inner ear hair cells, brain, and inactivated fibrosarcoma cells and whose relevance to nicotine signalingin primary lung cells is as yet uncharacterized.

Using a similar approach, as described for the analysis of CSC exposurein TABLE 13 and FIG. 37, the global effects of the exposure to the S9microsomal fraction were assessed by first identifying the subset of HVgenes that were correlated among all three treatment groups and thenassuming that the effect on these genes was due to the S9 microsomalfraction solely, since their expression characteristics did not changewhen the S9 microsomal fraction was combined with contact to a CSC. Asdescribed above, a Monte Carlo analysis was performed to define astatistically robust correlation coefficient unlikely to occur bychance. Using this threshold, the probability of identifying a genecorrelated in all three groups by chance was <1/total number of genesanalyzed, thereby confirming the high statistical specificity of thismethod.

As shown in TABLE 14, a set of 52 genes was identified and the probablefunction of these genes was assessed using PathwayAssist™ software (seeFIG. 39). Many of the genes appeared to have roles in modulatingapoptosis (e.g., AVEN, LIG1, PTEN, etc.) indicating that the predominantcellular response to chronic S9 microsomal fraction exposure is toactivate apoptotic programs. A second group of S9-modulated genesmodulates cellular surface chemistry, adhesion, and cellulardifferentiation (e.g., SIAT4B, KRT10, CDSN and EXT2). These resultsindicate that the inclusion of S9 microsomal fractions in toxicogeneticexperiments significantly modulates cellular physiology, which maycomplicate and bias the results assessing the effects of CSCs or anyother type of complex hydrocarbon mix requiring metabolic activation.

TABLE 14 Genes Specific for S9 Treatment GenBank accession no. Geneabbreviation Gene description NM_001303 COX10 COX10 homolog, cytochromec oxidase assembly protein AK056540 Homo sapiens cDNA FLJ31978, weaklysimilar to Probable hexosyltransferase NM_016013 LOC51103 CGI-65 proteinNM_031916 ASP AKAP-associated sperm protein NM_000947 PRIM2A Primase,polypeptide 2A (58 kD) NM_006927 SIAT4B Sialyltransferase 4B NM_006441MTHFS 5,10-methenyltetrahydrofolate synthetase NM_002699 POU3F1 POUdomain, class 3, transcription factor 1 NM_002954 RPS27A Ribosomalprotein S27a AK055508 FLJ11785 Rad50-interacting protein 1 NM_024636FLJ23153 Likely ortholog of mouse tumor necrosis-alpha-inducedadipose-related protein BC011231 Homo sapiens, Similar toangiotensinogen NM_007052 NOX1 NADPH oxidase 1 NM_000234 LIG1 Ligase I,DNA, ATP-dependent NM_032553 FKSG79 Putative purinergic receptorNM_000025 ADRB3 Adrenergic, beta-3-, receptor AF023203 Homo sapienshomeobox protein Og12 U50536 Human BRCA2 region, mRNA sequence CG011NM_000421 KRT10 Keratin 10 (epidermolytic hyperkeratosis; keratosispalmariset plantaris) NM_001264 CDSN Corneodesmosin NM_000355 TCN2Transcobalamin II; macrocytic anemia NM_000401 EXT2 Exostoses (multiple)2 NM_014214 IMPA2 Inositol(myo)-1(or 4)-monophosphatase 2 NM_003797 EEDEmbryonic ectoderm development AF319523 Homo sapiens RT-LI mRNA,complete sequence AF074331 PAPSS2 3′-phosphoadenosine 5′-phosphosulfatesynthase 2 AF189011 RNASE3L Putative ribonuclease III BC009752 Homosapiens, Similar to sex comb on midleg-like 1 (Drosophila) NM_000691ALDH3A1 Aldehyde dehydrogenase 3 family, memberA1 NM_006006 ZNF145 Zincfinger protein 145 (expressed in promyelocytic leukemia) NM_005831 NDP52Nuclear domain 10 protein L26584 RASGRF1 Ras protein-specific guaninenucleotide-releasing factor 1 NM_014182 HSPC160 HSPC160 proteinNM_004963 GUCY2C Guanylate cyclase 2C (heat stable enterotoxin receptor)AB023223 STXBP- Tomosyn TOM NM_018919 PCDHGA6 Protocadherin gammasubfamily A, 6 NM_002968 SALL1 Sal-like 1 (Drosophila) NM_003587 DDX16DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 16 AK024449 PP2135 PP2135protein AB034205 LUC7A Cisplatin resistance-associated overexpressedprotein BC011589 OSM Oncostatin M NM_006597 HSPA8 Heat shock 70 kDprotein 8 NM_004384 CSNK1G3 Casein kinase 1, gamma 3 AK057672 Homosapiens cDNA FLJ33110 fis NM_016344 PRO1900 PRO1900 protein NM_018651ZFP Zinc finger protein NM_004717 DGKI Diacylglycerol kinase, iotaNM_006479 PIR51 RAD51-interacting protein AK024250 Homo sapiens cDNAFLJ14188 fis NM_001382 DPAGT1 Dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 NM_020371 AVEN Cell deathregulator aven NM_006311 NCOR1 Nuclear receptor co-repressor 1

Discriminant Function Analysis (DFA) is a form of multivariate analysisthat identifies subsets of dependent variables that characterize asystem made up of related groups. In this kind of gene expressionanalysis, a linear equation is calculated, denoted a root, whose overallvalue is distinct for a given characterized group. Accordingly, DFAidentifies genes most characteristic of a given state. DFA analysis wasconducted on the genes that were correlated after CSC treatment but notcorrelated after S9 treatment, as described in the following example.

Example 11 Refined Analysis of CSC-Correlated Genes Using DiscriminantFunction Analysis (DFA)

The set of 40 genes that were correlated after CSC treatments (see Table13 and FIG. 37) but not correlated after S9 microsomal fractiontreatment were further analyzed using DFA. Of the 40 CSC-correlatedgenes, 11 were identified by DFA as being most highly distinct among CSCand S9 treated cells (Table 15). Interestingly, a significant number ofthese genes were associated with oncogenesis. For example, this gene setincluded 3 putative proto-oncogenes including (1) MAP2K5, theover-expression of which is associated with increased proliferative andinvasive potential of metastatic prostate cancer and is reported to be apotent survival molecule in APO-MCF-7 breast carcinoma cells; (2) DDIT3,a C/EBP transcriptional regulator involved in growth arrest induced byDNA damage that is a common breakpoint in human myxoid liposarcomas; and(3) BAG2, a BCL-2-binding apoptosis suppressor that is over-expressed inhuman cervical, breast and lung cancer cell lines. In addition, threeputative tumor suppressor genes were also identified in this gene set.These were FUS1, RASA1, and FPH2L1. FUS1 can inhibit tumor cell growthby inducing apoptosis, and was first identified in a search forpotential tumor suppressors within a critical homozygous deletion regionat 3p21.3 common in lung cancers. RASA1 as a key member of the GAP1family of GTPase-activating proteins plays a key role in the Rassignaling pathway. DPH2L1 is a BRCA1-induced gene that maps within aregion of 17p13.3, which is deleted in 80% of all ovarian epithelialmalignancies. DPH2L1 was identified by exon trapping in this region andwas implicated as a tumor suppressor as its expression is reduced orundetectable in ovarian tumors and tumor cell lines. In addition, anicotinic cholinergic receptor, CHRNA9, and two putative neural growthfactors, NxpH3, a neuropeptide-like neural signaling molecule, andNINJ2, a gene up-regulated in damaged nerve cells that upregulatesneurite outgrowth, were also identified in this gene set. The impact onneural growth factors is not surprising in light of the fact that manylung cancers express neuroendocrine features and are also stimulated byan autocrine/paracrine system of neuroendocrine peptide hormones.

A graphical representation of the DFA results for the three treatmentconditions at all time points was generated. The spatial organization ofthe elements in this representation provided a measure of the overallvariance among groups (see FIG. 40). The genes used for this analysiswere correlated in CSC exposed cells and not correlated in S9-treatedcells. A correlation coefficient of 0.8 was used as a threshold fordefining similarity. The expression of these genes should therefore besimilar in CSC-treated cells. Indeed the two CSC groups were moreclosely associated than either CSC group was to the S9 microsomalfraction-treated group. Of note, the samples from the CSC groups did notoverlap, indicating that the two CSC treatments elicit somewhat distinctresponses even in genes highly correlated in their behavior in each CSCgroup.

TABLE 15 Discriminant Function Analysis of CSC-Correlated Genes GenBankGene accession no. abbreviation Gene description M23326 TRDV3 T cellreceptor delta variable 3 NM_002757 MAP2K5 Mitogen-activated proteinkinase kinase 5 NM_004083 DDIT3 DNA-damage-inducible transcript 3NM_004282 BAG2 BCL2-associated athanogene 2 NM_007275 FUS1 Lung cancercandidate NM_003408 ZFP37 Zinc finger protein 37 homolog (mouse)NM_002046 GAPD Glyceraldehyde-3-phosphate dehydrogenase NM_017581 CHRNA9Cholinergic receptor, nicotinic BC015737 NINJ2 Ninjurin 2 AB032985 NXPH3Neurexophilin 3 NM_002890 RASA1 RAS p21 protein activator NM_001383DPH2L1 Diptheria toxin resistance protein

FIG. 41 shows the result of a functional analysis of the gene set inTABLE 14 using Pathway Assist. Not surprisingly, the major cellularprocesses affected by these genes were subset of the processes affectedby the parent gene set, as illustrated in FIG. 38.

Four post-treatment expression characteristics were established for eachgene on the array: (1) whether or not the gene was expressed abovebackground at each time-point; (2) whether or not the gene showedhypervariability (i.e. change greater than normal) of expression in one,two, or all three treatment conditions over the 12 h treatment period;(3) what was the specific pattern of gene expression over the 12 htreatment period; and (4) whether or not the gene expression pattern ineach condition correlated with its behavior under the two otherconditions from 0-12 h. Several interesting observations emerged fromthis analysis. Significantly, treatment of NHBE cells with CSCs from twoAmerican brands of cigarettes altered the expression of approximately3600 genes and ORFs (or 17% of the array) sometime during the 12-hourexposure (see FIGS. 1 and 2). These data provide evidence that due totheir chemical complexity and temporal requirement for metabolicactivation, CSCs should have a broad and dynamic effect on thehomeostatic transcriptome of the NHBE cell. In addition to thequantitative similarities in gene alterations induced by the differentCSCs, there were also qualitative similarities in that both CSCsaffected a large common block of genes, which is not surprising giventhe relatively comparable types of blended tobaccos used in mostAmerican cigarette brands.

Several approaches were employed to discriminate and cluster genes thatbecame hypervariable after CSC treatment so as to develop a robust andaccurate statistical estimate of functional significance for theseperturbations. For example, as shown in FIG. 38, CSCs affected networksof genes that intersect critical signaling pathways such as apoptosis,transcription, and cell cycle regulation, which are known to play keyroles in specific diseases such as cancer, chronic inflammation, andimpaired neural development, and which both epidemiological andfunctional studies conclude can be caused by chronic cigarette smoking.The relevance of these pathways to smoking-related diseases is furthersupported by a limited body of published data in which other cell typesor tissues exposed to either smoke, CSC, or a specific substance in CSC(e.g., benzo[a]pyrene, nicotine, etc.) were assessed using low-densityarrays (see Nadadur et al., Chest 121: 83S-84S, 2002; Nordskog et al.Cardiovasc Toxicol 3: 101-117, 2003; Zhang et al. Physiol Genomics 5:187-192, 2001; Gebel et al. Carcinogenesis 25:169-178, 2003).

The sensitivity and accuracy of the methodologies used herein toidentify genes impacted by CSCs was further shown by the fact that theset of HV genes in CSC-treated cells included many of the genes and/orgene families that have been identified using various global expressionanalyses (e.g., Serial Analysis of Gene Expression, DifferentialDisplay, and microarrays) and concluded to be of importance in thedevelopment and/or maintenance of lung cancers. These include erb-B2,matrix metalloproteinase 9 (MMP9), the heterogeneous nuclearribonucleoprotein (hnRNP) family, the Fus1 lung cancer candidate,glutathione S-transferase pi, the β-retinoic acid receptor, chromograninB, RAB5, death-associated protein kinase 1 (DAPK), various cancer/testisantigens [MAGE genes], and others. For the first time, however, thepresent disclosure demonstrates that expression of these genes isaltered in normal bronchial epithelial cells exposed to CSCs for only ashort period of time, which provides evidence that one or more of thesegenes are an early indicator of tobacco-related cellular damage. Inaddition, the data herein identify a large number of genes and genefamilies that had not yet been associated with the induction ormaintenance of pulmonary neoplasms or to other tobacco-related diseasesinvolving the cardiovascular and immune systems. Accordingly, many ofthe genes identified using the approaches described herein areparticularly useful biomarkers of the pathogenesis of these diseases.

The highly correlated expression characteristics of the CSC-impactedgenes shown in TABLE 7 and FIG. 38, for example, highlight several genesthat appear to play prominent roles in tobacco-related diseases. BothDPH2L1 and Fus1 are putative tumor suppressor genes associated withovarian and lung cancer, respectively. Fus1 is found at a homozygousdeleted region of chromosome 3p21 in lung tumors, and its forcedexpression in lung carcinoma cells suppresses cell growth in vitro andgrowth and metastases of tumors in vivo by mechanisms involvingG1-arrest and induction of apoptosis. The RASA1 is a component of theGAP1 family of GTPase-activating proteins, which can suppressproliferation signals by enhancing the weak intrinsic GTPase activity ofnormal RAS p21 protein and maintaining it in its inactive GDP-boundform. It is contemplated that Ras acts as a major nexus for multiplesignaling pathways that control a diverse range of functions, but manyof the subtleties of Ras functioning in individual cell types remainunclear. It is also though that Ras plays an important role in tumorcell survival. The MAP2K5 is a novel mitogen activated protein kinaseimplicated in the regulation of cell proliferation. Over-expression ofMAP2K5 can, in cooperation with other effectors, transform rodent cells,and function as a potent survival molecule in breast cancer cells.MAP2K5 represents a potential therapeutic target in prostate cancer asover-expression of MAP2K5 can induce proliferation, motility, andinvasion. Interestingly, MAP2K5 also dramatically up-regulates theexpression of matrix metalloproteinase-9 (MMP9) in prostate cancers. Asshown in TABLE 7, MMP9 was hypervariable in both CSC-treatment groups.The matrix metalloproteinases (MMPs) are a large family of extracellularmatrix degrading enzymes believed to play central roles in degradation,remodeling, and repair of basement membranes. Inappropriate orover-expression of these proteins appear to a critical determinant intumor invasion and metastasis of a number of neoplasms including thoseof the lung. For example, MMP9 potentiates pulmonary metastasisformation, and high serum levels of MMP9 in patients with non-small-celllung cancer (NSCLC) correlated with significantly shorter survival thanpatients with low serum levels of this protein.

In addition to a common set of affected genes, each individual CSC alsoaltered the expression of a relatively large gene set that was unique toeach CSC. That is, it was discovered that each tobacco smoke condensatewas associated with a unique genetic fingerprint. The impact on theseunique gene sets may be due to qualitative and/or quantitativedifferences in the constellation of chemical constituents in the twoCSCs. It is interesting to note that despite the fact that both Brand Aand Brand B are similar types of cigarettes (i.e., ‘full-flavor’) asdetermined by FTC criteria, there are measurable differences in thequantities of nicotine, tar, as well as, toxins and carcinogens betweenBrand-A and Brand-B cigarettes. It is contemplated that the differencesin one or more of these substances directly correlates with the observeddifferences in gene induction and level of expression. Moreover, it iscontemplated that each unique gene set affected by CSC-A and CSC-Bultimately influences different cellular pathways and results indifferent biological consequences.

Several basic assumptions of the emerging field of toxicogenomics arethat there are reasonable similarities in gene expression patternsinduced by multiple members of one specific class of toxicants, andsubtle differences in these gene expression patterns may distinguishdistinct chemical-specific ‘gene signatures’ of exposure (Afshari etal., Cancer Res 59: 4759-4760, 1999; Neumann et al. Biotechnol Adv 20:391-419, 2002). For the first time, the approaches described hereinprovide one with the ability to identify a unique genetic fingerprint orsignature for a plurality of tobacco products by contacting NHBE cellsor another cell type of the lung, mouth or oral cavity with a tobaccosmoke condensate or tobacco smoke from said plurality of tobaccoproducts, identifying the genes expressed as a result of the contact ineach individual tobacco product, as well as the level of expression ofeach, comparing the fingerprint or component thereof (e.g., a specificgene or set of genes or level of expression of a specific gene or set ofgenes) of the plurality of tobacco products that are being analyzed (orto a database containing genetic fingerprints of tobacco products),identifying differences in the fingerprint or component thereof betweenthe products that are being analyzed, and associating the difference inthe fingerprint or component thereof to an increased or decreased risk,proclivity, or potential to acquire a tobacco-related disease (e.g.,lung cancer).

Another significant discovery made in the experiments described above,as shown in FIG. 35, is that the majority of CSC-affected genes do notreturn to baseline within the 12-hour treatment period, especially forCSC-B-affected genes. This observation is not simply due to the factthat the cells were chronically exposed to the CSCs for the entire12-hours, as is discussed-infra. It is contemplated that many of theaffected genes require a significant amount of time to return tobaseline even after exposure is terminated. Accordingly, a currentpack-a-day smoker who averages >150 cigarette puffs/day may alter thehomeostatic expression of a large number of genes that cannot return toa baseline state during a typical day. This chronically perturbed state(either increased or decreased compared to baseline) of one or more ofthese genes may ultimately be etiologically involved in variouspathological states caused by exposure to cigarette smoke. Evidence ofthis is provided by the fact that in subjects who quit smoking there isboth short-term improvement in the functioning of a number of affectedorgan systems (e.g., lung, cardiovascular structures, kidneys, etc.) anda long-term decline in incidence and mortality from various diseasesaffecting these systems. Presumably, this reversal of smoking-relateddamage at the tissue and population levels reflects a correspondingreversal at a molecular and cellular level.

For example, chronic inflammatory processes in smokers play fundamentalroles in the pathogenesis of atherosclerosis, and increased plasma andtissue levels of several biomarkers associated with inflammation such asvarious cytokines (e.g., IL-1β, TNF-α), pro-atherogenic enzymes (e.g.,lipoprotein lipase) and cell adhesion molecules (e.g., VCAM-1) areassociated with future cardiovascular risk, while smoking cessationleads to decreased expression of many pro-inflammatory biomolecules anda concomitant reduction in cardiovascular risk. It is also possible thatthe altered expression of one or more genes in the habitual smokerbecomes attenuated with time as an adaptive response to the stress ofchronic activation, and this phenomenon may have unanticipated long-termbiological consequences for the smoker.

Another unexpected finding of this study was that the S9 metabolicenzyme fraction significantly influenced gene expression in NHBE cells.S9-exposed cells are traditionally considered a negative control fortoxicogenetic experiments performed to establish environmental andoccupational exposure guidelines. The fact that gene alterations wereobserved as early as 2 hours post-S9 exposure has interpretiveimplications for standard toxicological assays that routinely measurebiological and genetic effects of control and test substances after 4hours of exposure. This observation is particularly relevant as theglobal shift towards advanced genomic and proteomic technologiestransforms the field of toxicology from one relying on the induction ofgross genetic abnormalities such as mutations and structural/numericalchromosomal abnormalities to one where altered expression of panels ofgenes and proteins are used to determine risk to the human population.In order to clearly establish the potential toxicity or efficacy of anenvironmental substance, drug, or chemopreventive agent, it is importantto show that control substances or vehicles used in the methodologycause minimal disruption of the physiologically normal transcriptome.Furthermore, since S9 can induce a range of alterations in geneexpression levels independent of any test substance, it is possible thatone or more S9-induced effects can be synergistic or antagonistic withthe test substances. For example, FIG. 36 shows that many of the samegenes that are down-regulated in S9-treated cells are up-regulated inCSC-treated cells despite the fact that CSCs contain the sameconcentration of S9 enzymes. Alternatively, the effects of S9 can bemitigated by the test substance. Evidence for this is strongly supportedby the data, which shows that a number of genes whose steady-state mRNAlevel were found to be altered only by S9 were not found to be alteredwhen cells were exposed to S9 in context with either CSC-A or CSC-B. Inthis scenario, the direct effects of S9, which can be directly cytotoxicto cells in cultures, may be attenuated when sequestered and modifiedthrough contact with substances in CSCs.

Although the analysis of normal human bronchial epithelial cells (NHBEcells) contacted with tobacco smoke condensates, described above,provide several ways to identify the genes that are modulated inresponse to human exposure to tobacco smoke, another approach involvesanalysis of cells of the mouth, oral cavity, trachea, and lungs, eithernormal or immortalized cell lines (e.g., human bronchial cells (e.g.,BEP2D or 16HBE140 cells), human bronchial epithelial cells (e.g., HBECcells, 1198, or 1170-I cells), normal human bronchial epithelial cells(NHBE cells), BEAS cells (e.g., BEAS-2B), NCI-H292 cells, non-small celllung cancer (NSCLC) cells or human alveolar cells (e.g., H460, H1792,SK-MES-1, Calu, H292, H157, H1944, H596, H522, A549, and H226) tonguecells (e.g., CAL 27), and mouth cells (e.g., Ueda-1)), which arecontacted with cigarette smoke. Accordingly, as described in thefollowing example, several experiments were conducted to evaluate thegenes that were expressed, as well as the expression levels, when NHBEcells were exposed to tobacco smoke.

Example 12 Microarray Analysis in CS Experiments

Once the NHBE cells were contacted with tobacco smoke or with air (“mockexposure”), as described in Example 4, the cDNA of NHBE cells that wereeither mock exposed or tobacco smoke exposed was prepared for microarrayanalysis as follows. Cells were harvested for total RNA extraction aftereither mock or smoke treatment. The RNA from each Petri dish was usedfor a separate microarray chip, which resulted in a total of 18microarrays (ten from Experiment 1 and eight from Experiment 2). Themedium was aspirated and the dishes were rinsed twice with 1 mLprewarmed PBS per dish. After the second rinse, 1 mL of cold TRIzol®(Invitrogen Corp., Carlsbad, Calif.) was added to each dish. NHBE celllysates were prepared and the RNA was extracted according to themanufacturer's protocol. The RNA pellet was frozen and stored at −80° C.

Prior to cDNA synthesis, the RNA was resuspended indiethylpyrocarbonate-treated water. RNA integrity was assessed usingcapillary gel electrophoresis (Agilent BioAnalyzer, AgilentTechnologies, Palo Alto, Calif.) to determine the ratio of 28s:18s rRNAin each sample. A threshold of 1.0 was used to define samples ofsufficient quality and only these samples were used for microarraystudies. The RNA quality of all samples was extremely high with noratios less than 1.8. Fluorescently labeled cDNA was synthesized andpurified as previously described. (See Jarvis et al. Arthritis Res Ther,6: R15-R32, 2004, expressly incorporated by reference in its entirety).

A commercially available, genome-scale oligonucleotide librarycontaining gene-specific 70-mer oligonucleotides representing 21,329human genes was used for microarray production (QIAGEN Inc., Valencia,Calif.). Oligonucleotides were spotted onto Corning® UltraGAPS™amino-silane coated slides, which were then rehydrated with water vapor,snap dried at 90° C. Oligonucleotide DNAs were covalently fixed to thesurface of the glass using 300 mJ of ultraviolet radiation at a 254 nmwavelength. Unbound free amines on the glass surface were blocked for 15min with moderate agitation in a solution of 143 mM succinic anhydridedissolved in 1-methyl-2-pyrrolidinone, 20 mM sodium borate, pH 8.0.Slides were rinsed for 2 min in distilled water, immersed for 1 min in95% ethanol, and dried with a stream of nitrogen gas.

Hybridization was performed in an automated liquid delivery,air-vortexed, hybridization station for 9 hr at 58° C. under anoil-based cover slip (Ventana Medical Systems, Inc. Tucson, Ariz.).Microarrays were washed at a final stringency of 0.1×SSC. Microarrayswere scanned using a simultaneous dual color, 48-slide scanner (AgilentTechnologies). Fluorescent intensity was quantified using Imagene™software (BioDiscovery, Marina del Rey, Calif.).

Adjustment of expression levels in compared samples was performed aspreviously described. (See Dozmorov, et al. Bioinformatics., 19:2004-211, 2003; Knowlton, N., et al. Bioinformatics., 20: 3687-3690,2004; and Dozmorov, et al. Bioinformatics., 5:53, 2004, each of which isincorporated by reference in its entirety). To determine differentiallyexpressed genes, the analysis was confined to the set of genes that wereexpressed above background in at least one condition (i.e., 4 and/or 24hours post-exposure, CS-treated or mock-treated). For each experiment,replicates from each condition were averaged and genes that were under-or over-expressed (“modulated”) in response to tobacco smoke treatment(e.g., cigarette smoke (CS)) by 1.5-fold or more at either or both timepoints were identified. Genes exhibiting similar expression behavior inboth experiments were determined.

Quantitative Reverse Transcriptase PCR (qRT-PCR)

To determine the level of expression, RNA was reverse-transcribed usingan Omniscript RT™ kit according to manufacturer's instructions (Qiagen,Valencia, Calif.) and the resultant cDNA subsequently purified using theMontage PCR 96-well cleanup plate (Millipore, Billerica, Mass., USA).The qRT-PCR amplifications were performed on an ABI®PRISM 7700 sequencedetection system using SYBR®Green I dye assay chemistry. A 15 uL PCRreaction for each gene of interest was prepared consisting of 7.5 uL of2× SYBR®Green PCR mix (Applied Biosystems Inc., Foster City, Calif.),4.9 μl of H2O, 0.6 μl (30 pmoles) of gene-specific forward and reverseprimers, and 2 μl (1 ng) of cDNA template. All samples were run intriplicate with the appropriate single qRT-PCR controls (no reversetranscriptase and no template). Cycling conditions used for allamplifications were one cycle of 95° C. for 10 minutes and 40 cycles of95° C. for 15 seconds and 60° C. for 1 minute. Following the qRT-PCR,dissociation curve analysis was performed to determine if the desiredsingle gene product was produced.

Gene Expression Alterations Induced by CS Exposure

In order to determine the broad impact of a brief transient exposure tocigarette smoke (CS) on the transcriptome of NHBE cells, monolayercultures of NHBE cells were treated in logarithmic phase of growth for15 minutes with whole smoke from a leading representative brand ofAmerican cigarettes, and then assessed for global alterations in theirtranscriptome at 4 h and 24 h post-exposure. Furthermore, in an attemptto unambiguously define a set of genes consistently impacted by CS, thisexperiment was performed twice and then the focus was restricted to onlythose individual genes whose RNA levels similarly deviated by 1.5 foldor greater in the two experiments (either overexpressed orunderexpressed in response to CS treatment). By assessing global RNAchanges at 4 and 24 h post-exposure, the temporal relationships of thosegenes whose RNA levels were altered a) by 4 hours and that returned tobaseline by 24 hours; b) by 4 hours and did not return to baseline by 24hours; and c) only by 24 hours could be observed.

Approximately 10% of the 21,329 human genes represented on the arraywere expressed above background in mock-treated cells. This amount ofexpression presumably represents the typical transcriptome of unstressedNHBE cells in vitro, and agrees well with published data on the humanairway transcriptome of healthy nonsmokers. Interestingly, CS-treatedNHBE cells also expressed approximately 10% of the total genecomplement, suggesting that brief CS-exposure does not induce a majorquantitative reorganization of the normal transcriptome of lung cells.

Of the 21,329 genes on the array, a set of 364 genes exhibited similarchanges in expression level in both experiments (See TABLE 16). A subsetof 298 genes that were overexpressed 1.5-fold or more in bothexperiments was compared to mock-treated cells. Of this set of 298up-regulated genes, 184 were up-regulated exclusively at 4 h postcigarette smoke exposure, while 69 were up-regulated exclusively at 24 hpost-exposure, and 45 were up-regulated at both time points. The numberof genes that were under-expressed at least 1.5-fold in cells exposed tocigarette smoke was 66, with 35 down-regulated exclusively at 4 h postCS-exposure, 30 down-regulated exclusively at 24 h post-exposure, andone down-regulated at both time points. Further confirmation that theentire set of 364 up and down-regulated genes accurately reflect areliable genetic response to cigarette smoke exposure is evidenced bythe fact that a majority of the genes exhibited remarkably consistentexpression behaviors in both experiments.

TABLE 16 Genes Upregulated by Cigarette Smoke Fold Fold Increase atIncrease at Gene ID Gene Name Description 4 h 24 h NM_004261 SEP 15 15kDa selenoprotein 1.71 1.29 NM_000859 HMGCR 3-hydroxy-3-methylglutaryl-2.25 1.33 Coenzyme A reductase AK025736 HMGCS13-hydroxy-3-methylglutaryl- 1.02 1.63 Coenzyme A synthase 1 (soluble)NM_002526 NT5 5′ nucleotidase (CD73) 1.45 1.69 NM_001109 ADAM8 Adisintegrin and 1.17 2.72 metalloproteinase domain 8 NM_005891 ACAT2Acetyl-Coenzyme A 1.44 1.77 acetyltransferase 2 (acetoacetyl Coenzyme Athiolase) NM_006409 ARPC1A Actin related protein 2/3 complex, 2.01 1.79subunit 1A (41 kD) NM_018445 LOC55829 AD-015 protein 1.64 2.02 NM_001284AP3S1 Adaptor-related protein complex 2.18 1.27 3, sigma 1 subunitNM_000485 APRT Adenine 1.56 1.63 phosphoribosyltransferase NM_007002ADRM1 Adhesion regulating molecule 1 1.68 1.61 NM_006829 APM2 Adiposespecific 2 1.96 2.34 NM_001667 ARL2 ADP-ribosylation factor-like 2 2.060.80 NM_000693 ALDH1A3 Aldehyde dehydrogenase 1 family, 0.82 2.88 memberA3 NM_001635 AMPH Amphiphysin (Stiff-Mann 1.78 2.16 syndrome with breastcancer 128 kD autoantigen) NM_001657 AREG Amphiregulin (schwannoma- 1.960.33 derived growth factor) NM_001145 ANG Angiogenin, ribonuclease,RNase 1.61 1.10 A family, 5 NM_000700 ANXA1 Annexin A1 1.39 1.82NM_005139 ANXA3 Annexin A3 1.34 1.71 NM_001154 ANXA5 Annexin A5 2.402.43 NM_004034 ANXA7 Annexin A7 2.10 1.64 NM_016476 ANAPC11 APC11anaphase promoting 1.68 1.30 complex subunit 11 homolog (yeast)NM_016085 APR-3 Apoptosis related protein APR-3 1.44 0.84 NM_005721ACTR3 ARP3 actin-related protein 3 1.63 1.72 homolog (yeast) NM_017900AKIP aurora-A kinase interacting 2.07 5.18 protein M90355 BTF3L2 Basictranscription factor 3, like 2 1.87 1.47 NM_004281 BAG3 BCL2-associatedathanogene 3 3.85 1.58 NM_001196 BID BH3 interacting domain death 1.541.05 agonist NM_003860 BCRP1 Breakpoint cluster region protein, 1.991.52 uterine leiomyoma, 1-barrier to autointegration factor NM_014567BCAR1 Breast cancer anti-estrogen 1.00 1.88 resistance 1 NM_021096CACNA1I Calcium channel, voltage- 1.68 2.75 dependent, alpha 1I subunitNM_005186 CAPN1 Calpain 1, (mu/I) large subunit 1.62 1.11 NM_001750 CASTCalpastatin 1.47 1.76 NM_013376 SEI1 CDK4-binding protein p34SEI1 2.461.87 NM_015965 GRIM19 Cell death-regulatory protein 2.16 2.23 GRIM19NM_016041 F-LAN-1 CGI-101 protein 1.51 1.58 NM_016038 LOC51119 CGI-97protein 1.78 2.34 BC002971 CCT5 Chaperonin containing TCP1, 1.81 1.74subunit 5 (epsilon) NM_006429 CCT7 Chaperonin containing TCP1, 2.85 3.21subunit 7 (eta) NM_000647 CCR2 Chemokine (C-C motif) receptor 2 0.693.35 NM_012111 C14orf3 Chromosome 14 open reading 1.88 1.15 frame 3AK026450 C20orf162 Chromosome 20 open reading 1.16 1.49 frame 162NM_007096 CLTA Clathrin, light polypeptide (Lca) 1.96 2.01 BC010039 CLPCoactosin-like protein 1.54 1.24 NM_016451 COPB Coatomer proteincomplex, 1.82 1.79 subunit beta NM_007263 COPE Coatomer protein complex,2.58 2.98 subunit epsilon NM_004645 COIL Coilin 1.21 1.79 AL162070CORO1C Coronin, actin binding protein, 1C 2.00 1.59 NM_000389 CDKN1ACyclin-dependent kinase inhibitor 4.69 1.38 1A (p21, Cip1) NM_000099CST3 Cystatin C (amyloid angiopathy 2.11 1.54 and cerebral hemorrhage)NM_001554 CYR61 Cysteine-rich, angiogenic inducer, 2.44 0.67 61NM_007274 HBACH Cytosolic acyl coenzyme A 1.61 2.28 thioester hydrolaseNM_020189 DC6 DC6 protein 1.64 1.73 NM_004396 DDX5 DEAD/H(Asp-Glu-Ala-Asp/His) 2.01 4.10 box polypeptide 5 (RNA helicase, 68 kD)NM_001357 DDX9 DEAD/H (Asp-Glu-Ala-Asp/His) 1.44 1.53 box polypeptide 9(RNA helicase A, nuclear DNA helicase II- leukophysin AB040961 DTX2Deltex homolog 2 (Drosophila) 1.76 1.62 NM_007326 DIA1 Diaphorase (NADH)(cytochrome 1.84 2.06 b-5 reductase) NM_020548 DBI Diazepam bindinginhibitor 1.69 1.84 (GABA receptor modulator, acyl- Coenzyme A bindingprotein) NM_013253 DKK3 Dickkopf homolog 3 (Xenopus 1.64 0.84 laevis)NM_004405 DLX2 Distal-less homeo box 2 29.27 2.13 AL080156 DKFZP434J214DKFZP434J214 protein 2.97 1.43 NM_014045 DKFZP564C1940 DKFZP564C1940protein 1.79 1.73 NM_001539 DNAJA1 DnaJ (Hsp40) homolog, subfamily 2.111.85 A, member 1 NM_006145 DNAJB1 DnaJ (Hsp40) homolog, subfmaily 4.991.57 B, member 1 NM_004419 DUSP5 Dual specificity phosphatase 5 1.970.47 NM_001946 DUSP6 Dual specificity phosphatase 6 2.08 2.29 NM_014390p100 EBNA-2 co-activator (100 kD) 2.00 1.02 NM_005451 ENIGMA Enigma (LIMdomain protein) 1.21 2.34 NM_004092 ECHS1 Enoyl Coenzyme A hydratase,1.60 1.23 short chain, 1, mitochondrial NM_004431 EPHA2 EphA2 2.37 1.93NM_016357 EPLIN Epithelial protein lost in neoplasm 1.74 1.63 betaBF541376 ESTs, Weakly similar to FRHUL 2.71 4.50 ferritin light chain[H. sapiens] NM_003757 EIF3S2 Eukaryotic translation initiation 1.831.47 factor 3, subunit 2 (beta, 36 kD) NM_003755 EIF3S4 Eukaryotictranslation initiation 2.12 2.40 factor 3, subunit 4 (delta, 44 kD)NM_001417 EIF4B Eukaryotic translation initiation 2.33 2.41 factor 4BNM_004095 EIF4EBP1 Eukaryotic translation initiation 1.69 1.26 factor 4Ebinding protein 1 NM_005243 EWSR1 Ewing sarcoma breakpoint region 1 2.021.33 NM_005245 FAT FAT tumor suppressor homolog 1 1.87 0.77 (Drosophila)NM_004104 FASN Fatty acid synthase 1.24 1.60 AK054816 FTH1 Ferritin,heavy polypeptide 1 2.07 3.32 NM_001457 FLNB Filamin B, beta (actinbinding 1.05 1.90 protein 278) NM_014164 FXYD5 FXYD domain-containingion 1.24 1.67 transport regulator 5 AL365404 GPR108 G protein-coupledreceptor 108 2.00 1.17 NM_007278 GABARAP GABA(A) receptor-associated1.55 1.75 protein NM_001520 GTF3C1 General transcription factor IIIC,8.72 0.41 polypeptide 1 (alpha subunit, 220 kD) AK024486 GLTSCR2 Gliomatumor suppressor 2.63 1.85 candidate region gene 2 NM_001498 GCLCGlutamate-cysteine ligase, 8.96 1.40 catalytic subunit NM_002061 GCLMGlutamate-cysteine ligase, 2.85 1.56 modifier subunit NM_004446 EPRSGlutamyl-prolyl-tRNA synthetase 1.76 0.73 NM_002064 GLRX Glutaredoxin(thioltransferase) 3.12 2.31 NM_002083 GPX2 Glutathione peroxidase 23.71 9.99 (gastrointestinal) NM_000637 GSR Glutathione reductase 1.571.54 NM_002087 GRN Granulin 1.36 1.58 L24498 GADD45A Growth arrest andDNA-damage- 2.81 0.61 inducible, alpha NM_006644 HSP105B Heat shock 105kD 2.83 1.02 NM_002157 HSPE1 Heat shock 10 kD protein 1 1.92 1.34(chaperonin 10) NM_005345 HSPA1A Heat shock 70 kD protein 1A 5.77 1.30NM_006597 HSPA8 Heat shock 70 kD protein 8 1.48 4.56 NM_004134 HSPA9BHeat shock 70 kD protein 9B 2.23 1.39 (mortalin-2) NM_016292 TRAP1 Heatshock protein 75 1.57 1.05 NM_002133 HMOX1 Heme oxygenase (decycling) 155.83 2.81 NM_004712 HGS Hepatocyte growth factor- 1.21 1.64 regulatedtyrosine kinase substrate NM_001533 HNRPL Heterogeneous nuclear 1.500.89 ribonucleoprotein L AK057120 HMG1 High-mobility group (nonhistone1.72 0.79 chromosomal) protein 1 AF130111 HDAC3 Histone deacetylase 31.92 1.38 NM_001536 HRMT1L2 HMT1 hnRNP methyltransferase- 1.83 1.16 like2 (S. cerevisiae) AK023395 Homo sapiens cDNA FLJ13333 1.82 1.39 fis,clone OVARC1001828 AK054711 Homo sapiens cDNA FLJ30149 1.57 0.76 fis,clone BRACE2000280, weakly similar to MNN4 PROTEIN AK055071 Homo sapienscDNA FLJ30509 1.36 1.64 fis, clone BRAWH2000595 AK056736 Homo sapienscDNA FLJ32174 1.18 4.26 fis, clone PLACE6001064 AK024927 Homo sapienscDNA: FLJ21274 1.83 0.89 fis, clone COL01781 AK055564 Homo sapiens cDNA:FLJ22182 1.00 1.50 fis, clone HRC00953 AK026181 Homo sapiens cDNA:FLJ22528 4.30 1.72 fis, clone HRC12825 AK026902 Homo sapiens cDNA:FLJ23249 1.76 1.09 fis, clone COL04196 AL512727 Homo sapiens mRNA-cDNA2.01 2.48 DKFZp547P042 (from clone DKFZp547P042) AL117595 Homo sapiensmRNA-cDNA 2.71 1.30 DKFZp564C2063 (from clone DKFZp564C2063) AL050378Homo sapiens mRNA-cDNA 1.37 1.70 DKFZp586I1420 (from cloneDKFZp586I1420)-partial cds AF041429 Homo sapiens pRGR1 mRNA, 1.37 1.86partial cds AF118072 Homo sapiens PRO1716 mRNA, 5.32 19.31 complete cdsAF065241 Homo sapiens thioredoxin delta 3 1.20 1.80 (TXN delta 3) mRNA,partial cds BC010009 Homo sapiens, clone 1.49 1.93 IMAGE: 3355383, mRNA,partial cds BC011880 Homo sapiens, Similar to 1.07 1.65 hypotheticalprotein, MGC: 7764, clone MGC: 20548 IMAGE: 3607345, mRNA, compleBC017001 Homo sapiens, Similar to RIKEN 26.36 5.69 cDNA 1700127B04 gene,clone IMAGE: 4425440, mRNA, partial cds BC007307 Homo sapiens, Similarto zinc 1.89 1.59 finger protein 268, clone IMAGE: 3352268, mRNA,partial cds NM_014029 HSPC022 HSPC022 protein 1.33 3.77 NM_014047HSPC023 HSPC023 protein 1.64 1.98 AF161415 HSPC030 HSPC030 protein 4.271.52 NM_016099 LOC51125 HSPC041 protein 1.46 1.08 NM_014168 HSPC133HSPC133 protein 1.58 1.41 NM_014182 HSPC160 HSPC160 protein 1.28 2.58AL139112 Human DNA sequence from clone 1.88 2.68 GS1-103B18 onchromosome Xq27.1-27.3 Contains ESTs, STSs and GSSs. Con AL354915 HumanDNA sequence from clone 1.38 2.01 RP11-392A19 on chromosome 13. ContainsESTs, STSs and GSSs. Contains a NM_000182 HADHA Hydroxyacyl-Coenzyme A2.39 1.22 dehydrogenase/3-ketoacyl- Coenzyme A thiolase/enoyl- CoenzymeA hydratase (trif NM_016404 HSPC152 Hypothetical protein 1.59 1.30NM_016623 BM-009 Hypothetical protein 1.53 1.08 NM_015932 HSPC014Hypothetical protein 1.31 1.56 NM_015343 HSA011916 Hypothetical protein1.79 1.22 AF103803 H41 Hypothetical protein 1.63 2.00 NM_014886 YR-29Hypothetical protein 1.53 1.44 NM_018437 EDAG-1 Hypothetical proteinEDAG-1 1.46 1.94 NM_018306 FLJ11036 Hypothetical protein FLJ11036 2.072.12 NM_032813 FLJ14624 Hypothetical protein FLJ14624 1.80 2.88NM_022842 FLJ22969 Hypothetical protein FLJ22969 3.39 31.88 NM_031207HT036 Hypothetical protein HT036 1.26 2.55 NM_024508 MGC10796Hypothetical protein MGC10796 1.46 1.84 AK027859 MGC11266 Hypotheticalprotein MGC11266 2.46 2.14 NM_032771 MGC12217 Hypothetical proteinMGC12217 1.56 1.02 BC014850 MGC13071 Hypothetical protein MGC13071 1.741.98 NM_032899 MGC14128 Hypothetical protein MGC14128 1.15 6.78NM_024040 MGC2491 Hypothetical protein MGC2491 2.69 2.86 NM_024038MGC2803 Hypothetical protein MGC2803 1.59 1.48 NM_031943 IFP38 IFP382.11 1.95 NM_052815 IER3 Immediate early response 3 2.94 1.54 NM_016545IER5 Immediate early response 5 9.20 1.18 NM_005542 INSIG1 Insulininduced gene 1 2.02 2.62 NM_021999 ITM2B Integral membrane protein 2B1.84 1.06 NM_006147 IRF6 Interferon regulatory factor 6 2.30 1.09NM_000576 IL1B Interleukin 1, beta 0.98 3.03 Z17227 IL10RB Interleukin10 receptor, beta 1.74 1.68 NM_004508 IDI1 Isopentenyl-diphosphate delta1.89 2.68 isomerase NM_005354 JUND Jun D proto-oncogene 1.67 1.25NM_006854 KDELR2 KDEL (Lys-Asp-Glu-Leu) 2.03 1.42 endoplasmic reticulumprotein retention receptor 2 NM_000421 KRT10 Keratin 10 (epidermolytic1.87 1.68 hyperkeratosis-keratosis palmaris et plantaris) NM_000224KRT18 Keratin 18 1.22 1.81 NM_005555 KRT6B Keratin 6B 1.44 2.26NM_014815 KIAA0130 KIAA0130 gene product 1.31 4.73 NM_000899 KITLG KITligand 1.35 2.21 NM_001730 KLF5 Kruppel-like factor 5 (intestinal) 2.341.01 NM_003937 KYNU Kynureninase (L-kynurenine 3.31 3.29 hydrolase)NM_005558 LAD1 Ladinin 1 1.44 2.29 NM_016201 LCCP Leman coiled-coilprotein 1.89 1.09 NM_015925 LISCH7 Liver-specific bHLH-Zip 1.29 1.64transcription factor NM_014463 LSM3 Lsm3 protein 1.85 1.98 NM_004995MMP14 Matrix metalloproteinase 14 2.20 2.57 (membrane-inserted)NM_005916 MCM7 MCM7 minichromosome 1.60 1.07 maintenance deficient 7 (S.cerevisiae) NM_006428 MAAT1 Melanoma-associated antigen 1.99 1.43recognised by cytotoxic T lymphocytes NM_006636 MTHFD2 Methylenetetrahydrofolate 1.81 0.68 dehydrogenase (NAD+ dependent),methenyltetrahydrofolate cyclohydrolase NM_004528 MGST3 Microsomalglutathione S- 1.73 1.76 transferase 3 NM_022818 MAP1A/1BLC3Microtubule-associated proteins 2.18 0.95 1A/1B light chain 3 NM_014341MTCH1 Mitochondrial carrier homolog 1 1.81 1.69 NM_014161 MRPL18Mitochondrial ribosomal protein 3.58 1.63 L18 NM_021134 MRPL23Mitochondrial ribosomal protein 1.58 1.23 L23 NM_017446 MRPL39Mitochondrial ribosomal protein 1.74 1.13 L39 NM_021210 MUM2 MUM2protein 1.20 1.61 NM_004529 MLLT3 Myeloid/lymphoid or mixed- 1.15 2.41lineage leukemia (trithorax homolog, Drosophila)- translocated to, 3NM_033546 MLC-B Myosin regulatory light chain 1.95 1.89 AB032945 MYO5BMyosin VB 1.50 1.74 NM_017534 MYH2 Myosin, heavy polypeptide 2, 1.660.90 skeletal muscle, adult NM_002473 MYH9 Myosin, heavy polypeptide 9,non- 1.82 2.60 muscle NM_002356 MARCKS Myristoylated alanine-richprotein 0.22 2.70 kinase C substrate NM_000903 NQO1 NAD(P)Hdehydrogenase, 2.64 2.77 quinone 1 NM_004541 NDUFA1 NADH dehydrogenase1.27 1.88 (ubiquinone) 1 alpha subcomplex, 1 (7.5 kD, MWFE) NM_004548NDUFB10 NADH dehydrogenase 1.63 1.29 (ubiquinone) 1 beta subcomplex, 10(22 kD, PDSW) NM_004547 NDUFB4 NADH dehydrogenase 1.63 2.11 (ubiquinone)1 beta subcomplex, 4 (15 kD, B15) NM_002494 NDUFC1 NADH dehydrogenase1.70 1.17 (ubiquinone) 1, subcomplex unknown, 1 (6 kD, KFYI) NM_014328NESCA Nesca protein 1.52 1.23 BC010285 NET1 Neuroepithelial celltransforming 0.78 2.28 gene 1 NM_000271 NPC1 Niemann-Pick disease, typeC1 2.31 1.39 NM_006096 NDRG1 N-myc downstream regulated 1.50 1.95 gene 1NM_006164 NFE2L2 Nuclear factor (erythroid-derived 3.80 1.23 2)-like 2NM_003489 NRIP1 Nuclear receptor interacting 0.94 1.63 protein 1NM_017838 NOLA2 Nucleolar protein family A, 1.83 1.94 member 2 (H/ACAsmall nucleolar RNPs) NM_002820 PTHLH Parathyroid hormone-like hormone1.66 2.59 NM_020992 PDLIM1 PDZ and LIM domain 1 (elfin) 1.56 1.60NM_002574 PRDX1 Peroxiredoxin 1 1.68 1.80 NM_003713 PPAP2B Phosphatidicacid phosphatase 1.22 1.84 type 2B NM_002631 PGD Phosphogluconatedehydrogenase 4.37 23.25 NM_002632 PGF Placental growth factor, vascular3.61 1.79 endothelial growth factor-related protein NM_002658 PLAUPlasminogen activator, urokinase 1.69 1.78 NM_014287 PM5 PM5 protein1.55 1.54 NM_003819 PABPC4 Poly(A) binding protein, 1.62 1.25cytoplasmic 4 (inducible form) NM_000937 POLR2A Polymerase (RNA) II (DNA1.23 1.65 directed) polypeptide A (220 kD) NM_001198 PRDM1 PR domaincontaining 1, with 7.04 3.20 ZNF domain NM_002583 PAWR PRKC, apoptosis,WT1, regulator 1.96 1.50 NM_000917 P4HA1 Procollagen-proline, 2- 1.081.51 oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alphapolypeptide I NM_053024 PFN2 Profilin 2 1.73 1.17 AB051437 ProSAP2Proline rich synapse associated 2.30 1.25 protein 2 (rat) NM_002778 PSAPProsaposin (variant Gaucher 1.70 2.72 disease and variant metachromaticleukodystrophy) NM_000963 PTGS2 Prostaglandin-endoperoxide 6.51 0.98synthase 2 (prostaglandin G/H synthase and cyclooxygenase) BC013908PSMC1 Proteasome (prosome, macropain) 1.68 1.13 26S subunit, ATPase, 1NM_002806 PSMC6 Proteasome (prosome, macropain) 1.64 1.25 26S subunit,ATPase, 6 NM_002815 PSMD11 Proteasome (prosome, macropain) 1.77 1.35 26Ssubunit, non-ATPase, 11 NM_002812 PSMD8 Proteasome (prosome, macropain)2.17 3.03 26S subunit, non-ATPase, 8 NM_002797 PSMB5 Proteasome(prosome, macropain) 2.82 3.28 subunit, beta type, 5 NM_002799 PSMB7Proteasome (prosome, macropain) 1.36 1.74 subunit, beta type, 7NM_014330 PPP1R15A Protein phosphatase 1, regulatory 7.10 0.88(inhibitor) subunit 15A NM_004156 PPP2CB Protein phosphatase 2 (formerly1.67 1.11 2A), catalytic subunit, beta isoform NM_006808 SEC61B Proteintranslocation complex beta 1.44 1.57 NM_015714 G0S2 Putative lymphocyteG0/G1 0.90 6.31 switch gene BC012513 ARHE Ras homolog gene family, 2.390.99 member E NM_003979 RAI3 Retinoic acid induced 3 1.05 3.46 NM_001666ARHGAP4 Rho GTPase activating protein 4 2.49 1.96 NM_001033 RRM1Ribonucleotide reductase M1 1.54 0.87 polypeptide NM_002950 RPN1Ribophorin I 2.08 1.10 NM_001029 RPS26 Ribosomal protein S26 1.31 1.70NM_002953 RPS6KA1 Ribosomal protein S6 kinase, 1.65 2.00 90 kD,polypeptide 1 AB037819 RRBP1 Ribosome binding protein 1 3.68 2.68homolog 180 kD (dog) NM_014248 RBX1 Ring-box 1 1.30 2.13 NM_006743 RBM3RNA binding motif protein 3 2.01 1.74 NM_004902 RNPC2 RNA-binding region(RNP1, 1.61 0.75 RRM) containing 2 NM_000687 AHCY S-adenosylhomocysteine1.74 1.82 hydrolase AB051532 SEMA4B Sema domain, immunoglobulin 1.111.77 domain (Ig), transmembrane domain (TM) and short cytoplasmicdomain, (se NM_003900 SQSTM1 Sequestosome 1 3.34 2.82 NM_001085 SERPINA3Serine (or cysteine) proteinase 2.74 #DIV/0! inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 3 NM_030666 SERPINB1 Serine (orcysteine) proteinase 3.11 2.58 inhibitor, clade B (ovalbumin), member 1NM_000602 SERPINE1 Serine (or cysteine) proteinase 2.32 2.38 inhibitor,clade E (nexin, plasminogen activator inhibitor type 1), NM_015966SDBCAG84 Serologically defined breast 1.86 1.45 cancer antigen 84NM_006622 SNK Serum-inducible kinase 3.02 1.13 AB000462 SH3BP2SH3-domain binding protein 2 4.63 2.02 NM_003134 SRP14 Signalrecognition particle 14 kD 1.58 1.45 (homologous Alu RNA bindingprotein) NM_003145 SSR2 Signal sequence receptor, beta 1.64 1.79(translocon-associated protein beta) NM_007107 SSR3 Signal sequencereceptor, gamma 1.74 1.26 (translocon-associated protein gamma) AF395440HEJ1 Similar to DNAJ 2.50 1.94 NM_005870 SAP18 Sin3-associatedpolypeptide, 18 kD 1.50 1.21 NM_006109 SKB1 SKB1 homolog (S. pombe) 1.552.52 NM_015523 DKFZP566E144 Small fragment nuclease 2.04 1.55 NM_030981RAB1B Small GTP-binding protein 1.53 1.16 NM_006518 SPRR2C Smallproline-rich protein 2C 1.41 4.09 NM_005628 SLC1A5 Solute carrier family1 (neutral 1.87 0.82 amino acid transporter), member 5 NM_004207 SLC16A3Solute carrier family 16 1.56 2.65 (monocarboxylic acid transporters),member 3 NM_018976 SLC38A2 Solute carrier family 38, member 2 2.48 0.85NM_014331 SLC7A11 Solute carrier family 7, (cationic 2.40 0.73 aminoacid transporter, y+ system) member 11 NM_003130 SRI Sorcin 0.92 1.80NM_004599 SREBF2 Sterol regulatory element binding 1.47 1.03transcription factor 2 NM_006745 SC4MOL Sterol-C4-methyl oxidase-like1.68 1.82 NM_006918 SC5DL Sterol-C5-desaturase (ERG3 delta- 1.59 1.115-desaturase homolog, fungal)- like NM_006819 STIP1Stress-induced-phosphoprotein 1 2.88 2.34 (Hsp70/Hsp90-organizingprotein) NM_006704 SGT1 Suppressor of G2 allele of SKP1, 1.81 1.32 S.cerevisiae, homolog of NM_002999 SDC4 Syndecan 4 (amphigl ycan, 1.211.71 ryudocan) NM_006289 TLN1 Talin 1 1.53 1.59 NM_015641 TES Testisderived transcript (3 LIM 2.10 0.95 domains) NM_003217 TEGT Testisenhanced gene transcript 1.71 1.28 (BAX inhibitor 1) NM_003314 TTC1Tetratricopeptide repeat domain 1 1.68 2.06 NM_003329 TXN Thioredoxin1.39 2.24 NM_003330 TXNRD1 Thioredoxin reductase 1 7.66 2.72 NM_004238TRIP12 Thyroid hormone receptor 1.73 1.43 interactor 12 NM_006755 TALDO1Transaldolase 1 1.96 1.72 NM_003234 TFRC Transferrin receptor (p90,CD71) 1.51 3.15 NM_001064 TKT Transketolase (Wernicke- 1.60 1.44Korsakoff syndrome) NM_012459 TIMM8B Translocase of inner 1.32 1.57mitochondrial membrane 8 homolog B (yeast) NM_006470 TRIM16 Tripartitemotif-containing 16 1.57 1.53 NM_003449 TRIM26 Tripartitemotif-containing 26 1.39 2.55 NM_003289 TPM2 Tropomyosin 2 (beta) 2.131.79 NM_003404 YWHAB Tyrosine 3- 2.06 3.12 monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide NM_012321 LSM4 U6snRNA-associated Sm-like 1.61 0.95 protein M26880 UBC Ubiquitin C 1.731.07 NM_014501 E2-EPF Ubiquitin carrier protein 1.83 1.41 NM_003334 UBE1Ubiquitin-activating enzyme E1 1.91 1.67 (A1S9T and BN75 temperaturesensitivity complementing) AL110132 UBE2V1 Ubiquitin-conjugating enzymeE2 1.80 1.66 variant 1 BC007657 UBE2M Ubiquitin-conjugating enzyme 1.581.80 E2M (UBC12 homolog, yeast) NM_003364 UP Uridine phosphorylase 2.481.13 NM_003574 VAPA VAMP (vesicle-associated 1.85 1.71 membraneprotein)-associated protein A (33 kD) NM_012323 MAFF V-mafmusculoaponeurotic 1.71 0.72 fibrosarcoma oncogene homolog F (avian)NM_002359 MAFG V-maf musculoaponeurotic 1.85 1.41 fibrosarcoma oncogenehomolog G (avian) NM_002467 MYC V-myc myelocytomatosis viral 2.75 1.98oncogene homolog (avian) NM_006007 ZNF216 Zinc finger protein 216 2.011.29 NM_013360 ZNF222 Zinc finger protein 222 2.26 1.86 NM_004234 ZFP93Zinc finger protein 93 homolog 0.75 1.64 (mouse) Genes Downregulated byCigarette Smoke Gene ID Gene Name Description M4/S4 M24/S24 NM_006856ATF7 activating transcription factor 7 0.81 2.32 NM_001143 AMELYamelogenin, Y-linked 1.61 1.03 NM_001657 AREG amphiregulin (schwannoma-0.50 2.95 derived growth factor) AB053314 ALS2CR12 amyotrophic lateralsclerosis 2 2.01 1.12 (juvenile) chromosome region, candidate 12AK023086 CDNA FLJ13024 fis, clone 1.56 1.05 NT2RP3000865 BI820294 CDNAFLJ26296 fis, clone 1.69 0.89 DMC07192, highly similar to Ig kappa chainV-III region HAH precursor AK025253 CDNA FLJ42432 fis, clone 2.15 1.70BLADE2006412 NM_001271 CHD2 chromodomain helicase DNA 1.10 1.62 bindingprotein 2 NM_006589 C1orf2 chromosome 1 open reading 1.56 0.87 frame 2AK000796 C14orf129 chromosome 14 open reading 0.79 1.80 frame 129NM_001934 DLX4 distal-less homeobox 4 1.29 2.08 NM_005509 DMXL1 Dmx-like1 2.05 1.22 NM_004419 DUSP5 Dual specificity phosphatase 5 0.45 2.37NM_003494 DYSF dysferlin, limb girdle muscular 1.19 2.31 dystrophy 2B(autosomal recessive) NM_000145 FSHR follicle stimulating hormone 1.581.29 receptor NM_005708 GPC6 glypican 6 1.78 1.51 NM_002053 GBP1guanylate binding protein 1, 1.31 1.58 interferon-inducible, 67 kDaAB033063 HEG HEG homolog 0.88 1.97 NM_002129 HMGB2 High-mobility groupbox 2 0.69 2.78 NM_003542 HIST1H4F histone 1, H4f 1.57 1.92 NM_024598FLJ13154 hypothetical protein FLJ13154 0.81 1.67 NM_017933 FLJ20701hypothetical protein FLJ20701 1.37 2.03 NM_024037 MGC2603 hypotheticalprotein MGC2603 1.59 0.74 BC016840 MGC34695 hypothetical proteinMGC34695 0.99 2.33 AK027858 MGC4248 hypothetical protein MGC4248 1.530.92 NM_006903 PPA2 inorganic pyrophosphatase 2 0.63 1.64 NM_000526KRT14 keratin 14 (epidermolysis bullosa 1.09 2.13 simplex,Dowling-Meara, Koebner) NM_000424 KRT5 keratin 5 (epidermolysis bullosa1.48 1.86 simplex, Dowling- Meara/Kobner/Weber-Cockayne types) NM_005554KRT6A keratin 6A 1.53 1.17 NM_005556 KRT7 keratin 7 1.54 1.06 AK024583LOC400078 (LOC387888), 1.60 1.19 mRNA NM_005583 LYL1 lymphoblasticleukemia derived 1.73 1.17 sequence 1 AL137524 MRNA* cDNA 1.03 1.67DKFZp434H2218 (from clone DKFZp434H2218) AL117623 MRNA* cDNA 1.72 1.02DKFZp564O2364 (from clone DKFZp564O2364) NM_012334 MYO10 myosin X 2.081.13 AB007959 NHLH2 nescient helix loop helix 2 1.08 1.53 NM_002520 NPM1nucleophosmin (nucleolar 1.62 1.67 phosphoprotein B23, numatrin)NM_033014 OGN osteoglycin (osteoinductive 1.21 1.62 factor, mimecan)NM_024594 PANK3 pantothenate kinase 3 1.88 1.42 AB029015 PLCL2Phospholipase C-like 2 5.45 2.99 NM_018049 PLEKHJ1 pleckstrin homologydomain 2.48 1.68 containing, family J member 1 BC015542 PVR poliovirusreceptor 1.54 0.98 NM_018936 PCDHB2 protocadherin beta 2 1.64 1.02NM_000320 QDPR quinoid dihydropteridine 1.24 1.81 reductase NM_000456RAB5B RAB5B, member RAS oncogene 2.56 2.22 family NM_007273 REArepressor of estrogen receptor 0.83 1.51 activity NM_005978 S100A2 S100calcium binding protein A2 1.89 1.55 NM_016372 TPRA40 seventransmembrane domain 1.57 0.84 orphan receptor NM_006456 SIAT7Bsialyltransferase 7 ((alpha-N- 0.77 2.21 acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminide alpha-2,6- sialyltransferase) BNM_024624 SMC6L1 SMC6 structural maintenance of 1.74 1.71 chromosomes6-like 1 (yeast) AL353933 SLC22A15 solute carrier family 22 (organic1.85 1.07 cation transporter), member 15 AK027663 STC2 stanniocalcin 20.77 1.74 AK024451 DKFZp762C186 Tangerine 1.56 1.30 NM_005480 TROAPtrophinin associated protein 1.77 1.12 (tastin) NM_002466 MYBL2 v-mybmyeloblastosis viral 1.01 1.63 oncogene homolog (avian)-like 2 NM_006385ZNF211 Zinc finger protein 211 1.96 1.22 NM_005096 ZNF261 Zinc fingerprotein 261 1.68 1.53 NM_003430 ZNF91 Zinc finger protein 91 (HPF7, 1.471.53 HTF10) AC006033 1.52 1.21 AF111848 1.68 1.27 AK025272 8.36 4.55AL137077 2.59 1.28 L24498 0.31 1.58 NM_003590 2.06 1.03 NM_005774 1.721.29 NM_014111 1.53 2.49

A typical example is shown in FIG. 42, which compares the expression ofthe heat shock genes DnaJ (HSP40) A1/B1 at 4 and 24 h in mock-treatedand CS-treated cells in both experiments. The figure shows not only aconsistent temporal relationship in the two experiments with both genesbeing up-regulated by 4 hrs and then returning to baseline by 24 hrs,but also that there is a consistent relative level of expression betweenthe two genes (i.e., 4 hr expression levels of B1 exceed that of A1 inboth experiments).

Confirmation of Differential Expression by qRT-PCR

The relative expression levels of 6 genes that were determined bymicroarray analysis to be up-regulated in CS-treated NHBE cells werereassessed by quantitative PCR using RNA from samples taken at both 4and 24 hr. This gene set included: ferritin heavy polypeptide, nuclearfactor (erythroid-derived 2)-like 2, heat shock protein 70, hemeoxygenase, thioredoxin reductase, cyclooxygenase 2, and sequestosome 1.It was determined that beta-actin expression levels in the normalizedmicroarray data were nearly identical among all the CS and mock-treatedsamples, so this gene was used as an internal normalization standard inthese experiments. Quantitative PCR results were in strong qualitativeagreement with the microarray results, as all 6 genes were alsoup-regulated by CS when assessed by qRT-PCR. Moreover, the qRT-PCRresults recapitulated the general trends of expression at both 4 and 24hr that were observed by microarray (Table 17).

TABLE 17 Microarray data microarray Qpcr microarray qRT-PCR 4 hr/fold 4hr/fold 24 hr/fold 24 hr/fold Gene 1 change change change change FTH12.3 2.6 3.4 3.5 HSPA1A 16.1 25.1 2.4 5.0 NFE2L2 3.8 3.47 1.23 1.21TXNRD1 11.4 16.0 3.2 2.0 HMOX1 42.5 77.6 1.7 4.7 PTGS2 5.4 17.0 0 0SQSTM1 3.9 7.7 2.6 3.3

Since the wide range of gases, toxins, free radicals, and carcinogenspresent in tobacco smoke are believed to cause multiple types ofstructural and chemical damage, the NHBE cells that are exposed totobacco smoke would presumably have to mount an integrated biologicaland genetic response in an attempt to prioritize and attenuate thisdamage. In an effort to understand the type of response mounted by theNHBE cells after cigarette smoke exposure, several databases wereanalyzed and genes that were identified as being over-expressed orunder-expressed in response to exposure to cigarette smoke were groupedaccording to functional similarities. The following example describesthis effort in greater detail.

Example 13 Functional Grouping of Genes Modulated in Response to CSExposure

Information from the Gene Ontology (GO) Consortium and from thescientific literature was used to categorize the genes identified asbeing modulated (i.e., over-expressed or under-expressed) in response tocigarette smoke exposure. Of the genes up-regulated by CS exposure thathave known functions (235 out of 298 genes), four major groups offunctionally related genes were identified (Table 11). These four groupscollectively represent a large proportion (45%; 105 out of 235 genes) ofthe differentially expressed genes with known function, indicating thatthese genes are involved in biological pathways that are highlyresponsive to CS-induced damage. In contrast, although 42 of the 66genes that were under-expressed in response to CS have known functions,they reflected multiple biological processes without a clear dominanceof specific function. As can be seen in Table 11, the predominantpathways highlighted by the over-expressed gene set indicate that thecell is responding to a sudden increase in oxidative stress and theconcentration of misfolded or damaged proteins, while simultaneouslyattempting to modulate its cell cycle and apoptotic controls.Unexpectedly, it was also observed that a proportionally large group ofCS-responsive genes are related to the metabolism and cellulartrafficking of cholesterol.

TABLE 18 Fold Fold Increase Increase Gene ID Symbol Description at 4 hat 24 h RESPONSE TO OXIDATIVE STRESS BF541376 FTL ESTs, Weakly similarto FRHUL 2.71 4.50 ferritin light chain [H. sapiens] AK054816 FTH1Ferritin, heavy polypeptide 1 2.07 3.32 NM_001498 GCLCGlutamate-cysteine ligase, catalytic 8.96 1.40 subunit NM_002061 GCLMGlutamate-cysteine ligase, modifier 2.85 1.56 subunit NM_002064 GLRXGlutaredoxin (thioltransferase) 3.12 2.31 NM_002083 GPX2 Glutathioneperoxidase 2 3.71 9.99 (gastrointestinal) NM_000637 GSR Glutathionereductase 1.57 1.54 NM_002133 HMOX1 Heme oxygenase (decycling) 1 55.832.81 NM_005354 JUND Jun D proto-oncogene 1.67 1.25 NM_004528 MGST3Microsomal glutathione S- 1.73 1.76 transferase 3 NM_000903 NQO1 NAD(P)Hdehydrogenase, quinone 1 2.64 2.77 NM_006096 NDRG1 N-myc downstreamregulated gene 1 1.50 1.95 NM_006164 NFE2L2 Nuclear factor(erythroid-derived 2)- 3.80 1.23 like 2 NM_020992 PDLIM1 PDZ and LIMdomain 1 (elfin) 1.56 1.60 NM_002574 PRDX1 Peroxiredoxin 1 1.68 1.80NM_000687 AHCY S-adenosylhomocysteine hydrolase 1.74 1.82 NM_003329 TXNThioredoxin 1.39 2.24 NM_003330 TXNRD1 Thioredoxin reductase 1 7.66 2.72NM_012323 MAFF V-maf musculoaponeurotic 1.71 0.72 fibrosarcoma oncogenehomolog F (avian) NM_002359 MAFG V-maf musculoaponeurotic 1.85 1.41fibrosarcoma oncogene homolog G (avian) CELLGROWTH/PROLIFERATION/APOPTOSIS NM_001657 AREG Amphiregulin(schwannoma-derived 1.96 0.33 growth factor) NM_016085 APR-3 Apoptosisrelated protein APR-3 1.44 0.84 NM_017900 AKIP aurora-A kinaseinteracting protein 2.07 5.18 NM_001196 BID BH3 interacting domain death1.54 1.05 agonist NM_005186 CAPN1 Calpain 1, (mu/I) large subunit 1.621.11 NM_013376 SEI1 CDK4-binding protein p34SEI1 2.46 1.87 NM_015965GRIM19 Cell death-regulatory protein 2.16 2.23 GRIM19 NM_001554 CYR61Cysteine-rich, angiogenic inducer, 2.44 0.67 61 NM_004396 DDX5 DEAD/H(Asp-Glu-Ala-Asp/His) 2.01 4.10 box polypeptide 5 (RNA helicase, 68 kD)NM_013253 DKK3 Dickkopf homolog 3 (Xenopus 1.64 0.84 laevis) NM_004419DUSP5 Dual specificity phosphatase 5 1.97 0.47 NM_001946 DUSP6 Dualspecificity phosphatase 6 2.08 2.29 NM_004431 EPHA2 EphA2 2.37 1.93NM_005245 FAT FAT tumor suppressor homolog 1 1.87 0.77 (Drosophila)NM_002087 GRN Granulin 1.36 1.58 L24498 GADD45A Growth arrest andDNA-damage- 2.81 0.61 inducible, alpha AF130111 HDAC3 Histonedeacetylase 3 1.92 1.38 AF103803 H41 Hypothetical protein 1.63 2.00NM_052815 IER3 Immediate early response 3 2.94 1.54 NM_016545 IER5Immediate early response 5 9.20 1.18 NM_000576 IL1B Interleukin 1, beta0.98 3.03 NM_001730 KLF5 Kruppel-like factor 5 (intestinal) 2.34 1.01NM_004529 MLLT3 Myeloid/lymphoid or mixed-lineage 1.15 2.41 leukemia(trithorax homolog, Drosophila)-translocated to, 3 NM_002632 PGFPlacental growth factor, vascular 3.61 1.79 endothelial growthfactor-related protein NM_002658 PLAU Plasminogen activator, urokinase1.69 1.78 NM_001198 PRDM1 PR domain containing 1, with ZNF 7.04 3.20domain NM_002583 PAWR PRKC, apoptosis, WT1, regulator 1.96 1.50NM_014330 PPP1R15A Protein phosphatase 1, regulatory 7.10 0.88(inhibitor) subunit 15A NM_015714 G0S2 Putative lymphocyte G0/G1 switch0.90 6.31 gene NM_001666 ARHGAP4 Rho GTPase activating protein 4 2.491.96 NM_006622 SNK Serum-inducible kinase 3.02 1.13 NM_006109 SKB1 SKB1homolog (S. pombe) 1.55 2.52 NM_006704 SGT1 Suppressor of G2 allele ofSKP1, S. cerevisiae, 1.81 1.32 homolog of NM_003217 TEGT Testis enhancedgene transcript 1.71 1.28 (BAX inhibitor 1) NM_002467 MYC V-mycmyelocytomatosis viral 2.75 1.98 oncogene homolog (avian)UBIQUITINATION/PROTEIN TURNOVER/HEAT SHOCK NM_001109 ADAM8 A disintegrinand metalloproteinase 1.17 2.72 domain 8 NM_004281 BAG3 BCL2-associatedathanogene 3 3.85 1.58 BC002971 CCT5 Chaperonin containing TCP1, 1.811.74 subunit 5 (epsilon) NM_006429 CCT7 Chaperonin containing TCP1, 2.853.21 subunit 7 (eta) NM_007278 GABARAP GABA(A) receptor-associated 1.551.75 protein NM_001539 DNAJA1 DnaJ (Hsp40) homolog, subfamily 2.11 1.85A, member 1 NM_006145 DNAJB1 DnaJ (Hsp40) homolog, subfmaily 4.99 1.57B, member 1 AF395440 HEJ1 Similar to DNAJ 2.50 1.94 NM_006644 HSP105BHeat shock 105 kD 2.83 1.02 NM_002157 HSPE1 Heat shock 10 kD protein 11.92 1.34 (chaperonin 10) NM_005345 HSPA1A Heat shock 70 kD protein 1A5.77 1.30 NM_006597 HSPA8 Heat shock 70 kD protein 8 1.48 4.56 NM_004134HSPA9B Heat shock 70 kD protein 9B 2.23 1.39 (mortalin-2) NM_016292TRAP1 Heat shock protein 75 1.57 1.05 NM_006819 STIP1Stress-induced-phosphoprotein 1 2.88 2.34 (Hsp70/Hsp90-organizingprotein) NM_004995 MMP14 Matrix metalloproteinase 14 2.20 2.57(membrane-inserted) BC013908 PSMC1 Proteasome (prosome, macropain) 1.681.13 26S subunit, ATPase, 1 NM_002806 PSMC6 Proteasome (prosome,macropain) 1.64 1.25 26S subunit, ATPase, 6 NM_002815 PSMD11 Proteasome(prosome, macropain) 1.77 1.35 26S subunit, non-ATPase, 11 NM_002812PSMD8 Proteasome (prosome, macropain) 2.17 3.03 26S subunit, non-ATPase,8 NM_002797 PSMB5 Proteasome (prosome, macropain) 2.82 3.28 subunit,beta type, 5 NM_002799 PSMB7 Proteasome (prosome, macropain) 1.36 1.74subunit, beta type, 7 NM_006808 SEC61B Protein translocation complexbeta 1.44 1.57 NM_014248 RBX1 Ring-box 1 1.30 2.13 NM_003900 SQSTM1Sequestosome 1 3.34 2.82 NM_003134 SRP14 Signal recognition particle 14kD 1.58 1.45 (homologous Alu RNA binding protein) NM_003314 TTC1Tetratricopeptide repeat domain 1 1.68 2.06 NM_004238 TRIP12 Thyroidhormone receptor interactor 1.73 1.43 12 M26880 UBC Ubiquitin C 1.731.07 NM_014501 E2-EPF Ubiquitin carrier protein 1.83 1.41 NM_003334 UBE1Ubiquitin-activating enzyme E1 1.91 1.67 (A1S9T and BN75 temperaturesensitivity complementing) AL110132 UBE2V1 Ubiquitin-conjugating enzymeE2 1.80 1.66 variant 1 BC007657 UBE2M Ubiquitin-conjugating enzyme E2M1.58 1.80 (UBC12 homolog, yeast) NM_000859 HMGCR3-hydroxy-3-methylglutaryl- 2.25 1.33 Coenzyme A reductase AK025736HMGCS1 3-hydroxy-3-methylglutaryl- 1.02 1.63 Coenzyme A synthase 1(soluble) CHOLESTEROL/LIPID METABOLISM NM_005891 ACAT2 Acetyl-Coenzyme A1.44 1.77 acetyltransferase 2 (acetoacetyl Coenzyme A thiolase)NM_000700 ANXA1 Annexin A1 1.39 1.82 NM_007274 HBACH Cytosolic acylcoenzyme A thioester 1.61 2.28 hydrolase NM_020548 DBI Diazepam bindinginhibitor (GABA 1.69 1.84 receptor modulator, acyl-Coenzyme A bindingprotein) NM_004092 ECHS1 Enoyl Coenzyme A hydratase, short 1.60 1.23chain, 1, mitochondrial NM_004104 FASN Fatty acid synthase 1.24 1.60NM_000182 HADHA Hydroxyacyl-Coenzyme A 2.39 1.22dehydrogenase/3-ketoacyl- Coenzyme A thiolase/enoyl- Coenzyme Ahydratase NM_005542 INSIG1 Insulin induced gene 1 2.02 2.62 NM_004508IDI1 Isopentenyl-diphosphate delta 1.89 2.68 isomerase NM_000271 NPC1Niemann-Pick disease, type C1 2.31 1.39 NM_003713 PPAP2B Phosphatidicacid phosphatase type 1.22 1.84 2B NM_002778 PSAP Prosaposin (variantGaucher disease 1.70 2.72 and variant metachromatic leukodystrophy)NM_004599 SREBF2 Sterol regulatory element binding 1.47 1.03transcription factor 2 NM_006745 SC4MOL Sterol-C4-methyl oxidase-like1.68 1.82 NM_006918 SC5DL Sterol-C5-desaturase (ERG3 delta- 1.59 1.115-desaturase homolog, fungal)-like

In order to visualize any underlying temporal expression patterns amongthese four functional classes a hierarchical clustering of the genes wasmade (see FIG. 43). This cluster analysis of the expression data showstwo important points: 1) that the four conditions (4 & 24 h mock-treatedand 4 & 24 h CS-treated) are clearly distinguishable by these functionalgroups of genes; and 2) that the expression of the specific genes in thefour functional groups do not have strong temporal relationships (i.e.they do not overwhelmingly cluster within either the 0-4 hours or 4-24hour time frame). However, it is clear from FIG. 43 that the majority ofthe CS-responsive genes in these functional groups exhibit a higherexpression at 4 h post-exposure than at 24 h. Since the cells weretreated for only 15 minutes and then analyzed for a change in geneexpression after 4 and 24 hrs, the decrease in expression for many ofthese genes by 24 hrs indicates that the cell is attempting to “reset”its transcriptome to pre-exposure levels, which would not be anunexpected response to a transient insult. However, the fact that theexpression of many of these genes remains increased over pre-exposurelevels for up to 24 hrs also indicates that the biological ramificationsof CS-exposure can affect the cell for a long period of time afterexposure to tobacco smoke is terminated. Accordingly, it is plausiblethat many of these genes may not return to homeostatic baseline in ahabitual smoker, which may have unforeseen pathological consequences.

A notable exception to most of the genes shown in FIG. 43 and TABLE 18,whose expression remain elevated up to 24 hrs post-exposure, is a largeblock of genes in the protein damage/turnover group, and which encodeprimarily heat shock and heat shock-associated proteins. The expressionof these heat shock related genes is dramatically elevated at 4 hrs butreturns to baseline by 24 hrs, indicating that the processes that engageand clear a buildup of CS-induced damaged and dysfunctional proteins arerapid. Finally, there are a small subset of genes whose expressionlevels are higher at 24 h than at 4 h, including ferritin, NADHdehydrogenase, peroxiredoxin 1, and glutathione peroxidase. Since eachof these genes is involved in redox reactions, it could signify thatoxidative stress caused by CS induces long-lived perturbations to redoxhomeostasis.

The four major functional groups of genes listed in Table 18 and shownin FIG. 43 show a well-organized attempt by the NHBE cell to attenuatethe damage caused by exposure to tobacco smoke. This type of coordinatedresponse provides evidence that functionally related blocks of genes aretranscriptionally regulated by the same or similar transcriptionalactivators. In the full set of 298 genes up-regulated by CS (see TABLE16), there are 21 genes with gene products that function astranscriptional regulators, including v-myc, interferon regulatoryfactor 6, eukaryotic translation initiation factor 4B, Kruppel-likefactor 5, sterol regulatory element binding transcription factor 2(SREB2), and Nuclear factor (erythroid-derived 2)-like 2 (NRF2). NRF2 isof particular interest in this regard since studies of NRF2-knockoutmice show that this transcription factor activates over 200 genes inseveral functional classes with the two most predominant being oxidativestress response and protein turnover (Kwak et al., J. Biol. Chem. (2003)278:8135-8145). As shown in Table 18, both of these classes of genes aredisproportionately activated by exposure of NHBE cells to tobacco smoke.Specifically, of the 105 genes presented in Table 18, 33 are known to beunder transcriptional control of NRF2, or to act as cofactors forNRF2-regulated transcription (see FIG. 43).

In addition, it has been shown that the short-term exposure of mice tocigarette smoke results in the induction of a set of 46 protectivegenes, all of which are under the control of NRF2 (Rangasamy et al., J.Clin. Invest. (2004) 114:1248-1259). In concordance with thisobservation, the data show that despite only brief exposure cells to CSin vitro, the RNA levels of 19 human homologues of these 46 mouse genes(41%) are similarly induced, indicating that the CS-related molecularevents occurring in vitro are very similar to those observed in vivo.This set of CS-induced genes in both the mouse and NHBE cells includesthose responsive to oxidative stress (heme oxygenase, phosphogluconatedehydrogenase, thioredoxin reductase, glutathione pathway genes,NADPH:quinone reductase), protein damage (HSP40, mortalin, GADD45), andprotein turnover (ubiquitin C, proteasome subunits, sequestosome).

The fact that cigarette smoke, as well as various constituents ofcigarette smoke, can cause disruptions to the genome, transcriptome, andproteome, allows one to develop a set of relevant biomarkers that areuseful for monitoring exposure to tobacco toxins, detectingpre-malignant disease, improving diagnosis and prognosis of currentdisease, developing new treatment options, and testing risk reductionstrategies for current and former smokers. A number of studies assessingthe clinical usefulness of alterations in global gene and proteinexpression patterns in malignant and normal human lung tissues haverecently shown that quantitative and/or qualitative changes in a smallnumber of expressed genes and proteins, in combination with standardclinicopathological variables, may have prognostic and/or diagnosticpotential in patients with tobacco-related diseases. Thus, elucidatingthe various molecular, genetic, and cellular dysfunctions induced bytobacco smoke may not only reveal a useful set of tobacco-specificbiomarkers, but also result in a detailed mechanistic understanding ofhow chronic tobacco exposure causes disease.

In more embodiments, a second tobacco product (e.g., a cigarette) iscompared to a first tobacco product (e.g., a cigarette) using themethods above so as to identify which of the two tobacco products isless likely to contribute to a tobacco-related disease. For example, afirst population of isolated human cells of the mouth, tongue, oralcavity, or lungs (e.g., NHBE cells), is contacted with a CS from a firsttobacco product (e.g., a “reduced risk full flavor” cigarette) in anamount and for a time sufficient to modulate expression of one or moregenes or to modify a gene product, and identification of the genes thatare modulated or modified gene product (e.g., phosphorylated) or thelevel or amount of gene expression or modification can be determinedusing any technique available that analyzes transcription (e.g.,microarray, genechip, qRT-PCR or hybridization), protein production(e.g., ELISA, Western blot, or other antibody detection techniques),modifications of proteins (e.g., oxidation or phosphorylation), or theappearance or disappearance of metabolites associated with genes thatare modulated in response to exposure to CS (e.g., cysteine,glutathione, fragments of proteins or lipids or fatty acids). A secondpopulation of isolated human cells of the mouth, tongue, oral cavity, orlungs (e.g., NHBE cells), preferably the same type of cell as used inthe analysis of the first tobacco product, is also contacted with a CSfrom a second tobacco product (e.g., a cigarette) in an amount and for atime sufficient to modulate expression of one or more genes or to modifya gene product. Identification of a gene that is modulated or modifiedgene product (e.g., phosphorylated) or the level or amount of geneexpression or modification can be accomplished using any techniqueavailable that analyzes transcription (e.g., microarray, genechip,qRT-PCR or hybridization), protein production (e.g., ELISA, Westernblot, or other antibody detection techniques), modifications of proteins(e.g., oxidation or phosphorylation), or the appearance or disappearanceof metabolites associated with genes that are modulated in response toexposure to CS (e.g., cysteine, glutathione, fragments of proteins orlipids or fatty acids).

The data obtained from the analysis of the first tobacco product can becompared to the data obtained from the analysis of the second tobaccoproduct so as to identify, for example, a gene(s) that are induced inresponse to exposure to the first tobacco product but not the secondtobacco product or vice versa. Additionally, the comparison will revealthat the level of expression of one or more genes induced by bothtobacco products differs with respect to the two tobacco products orthat the first product has more, less, or no modification of aparticular gene product (e.g., phosphorylation), as compared to thesecond tobacco product or vice versa. These data (e.g., the types ofgenes expressed, the amount of expression, and modification) allow oneto develop a profile for each tobacco product analyzed (in this exampleonly two products are being compared but a plurality of products can becompared using the same approach). These tobacco product profiles can berecorded on a computer readable media and databases containing thisinformation can be created. Once a gene is identified, it can beanalyzed using PathwayAssist™ software (Stratagene, La Jolla, Calif.),Genespring (version 7.2, Agilent Technologies), or other similarsoftware so as to determine whether the gene contributes to atobacco-related disease.

By analyzing the differences between the tobacco products analyzed,(e.g., the types of genes expressed, the amount of expression, andmodifications), one can identify a tobacco product that has lesspotential to contribute to a tobacco related disease or that, forexample, a first tobacco product has a reduced risk to contribute to atobacco-related disease, as compared to a second tobacco product or viceversa. By one technique, for example, a tobacco product that is lesslikely to contribute to a tobacco-related disease is identified becauseit induces fewer genes associated with a tobacco-related disease. Arelated approach (using CSC) was employed to identify a tobacco productas having a reduced potential to contribute to a tobacco-relateddisease, as compared to a second tobacco product. (See Examples 4-6).

The methods provided herein can be used not only to identify a tobaccoproduct that has a reduced potential to contribute to a tobacco-relateddisease, as compared to a second tobacco product, but also to developtobacco products that have a reduced potential to contribute to atobacco-related disease, as compared to a second tobacco product. Thatis, by coordinating techniques (e.g., chemical or genetic modification)to modulate expression of genes that produce various components intobacco with the analytical methods disclosed herein, one can rapidlydetermine whether the modulation of a particular gene that produces aparticular component in tobacco results in a modulation of a gene inhuman cells (e.g., NHBE cells) that results in a reduced potential tocontribute to a tobacco-related disease, as compared to the tobaccoprior to modulation of component-producing gene. The section belowdescribes these embodiments in greater detail.

Epidemiological Determinations

In still more embodiments, cells of the mouth, oral cavity, trachea, orlung (e.g., NHBE cells) from a plurality of individuals, preferably thesame cell type, are independently contacted with a tobacco composition(e.g., CS) in an amount and for a time sufficient to induce damage ofcellular genetic material or modulate cell homeostasis. The fact thatCS, as well as various constituents of CS, can cause disruptions to thecell allows one to develop a set of relevant biomarkers that are usefulfor monitoring exposure to tobacco toxins, detecting pre-malignantdisease, improving diagnosis and prognosis of current disease,developing new treatment options, testing chemopreventive compounds, andtesting risk reduction strategies for current and former smokers.Accordingly, also provided herein are methods of detecting pre-malignantdisease, improving diagnosis and prognosis of current disease,developing new treatment options, testing chemopreventive compounds, andtesting risk reduction strategies for current and former smokers bydetermining the amount of induction of damage of cellular geneticmaterial or modulation of cell homeostasis to the cells of a smoker orother tobacco consumer or a subject exposed to a tobacco composition.The cells of different individuals can respond differently to tobaccocompositions and thereby have different levels of risk of developing atobacco-related disease. The methods provided herein for determining amodulation of cell homeostasis, or determining a marker indicative ofmodulation of cell homeostatis, such as the methods of determining amodulation of gene expression (e.g., transcriptome or proteomemodulation), or determining the amount of induction of damage ofcellular genetic material in cells contacted with a tobacco compositioncan be used to assess a subject's level of risk of developing atobacco-related disease. Such methods can be generally performed inaccordance with the methods provided herein, where the cells of thesubject can be first contacted with smoke from the tobacco product invivo (e.g., by the subject smoking a cigarette or side-stream smokeexposure), and then the cells can be harvested using known methods(e.g., lung lavage or cheek swab); alternatively, the cells of a subjectcan be first harvested and optionally cultured, and then contacted withsmoke from the tobacco product in accordance with the methods providedherein. Provided below are non-limiting exemplary methods for testingtobaccos, tobacco products, compounds and the like; it is understoodthat any of the methods provided herein for monitoring a modulation ofcell homeostasis can be used in the examples provided below.

In one example, primary cultures of lung cells, bronchial cells, cellsof the mouth, pharynx, larynx, and tongue can be generated from anindividual to be tested and these cells are be contacted with a tobaccocomposition (e.g., CS from a tobacco product) so as to elucidate theindividuals proclivity to acquire a tobacco related disease. Certainpatterns of amount of induction of damage of cellular genetic materialor modulation of cell homeostasis to tobacco compositions can beassociated with individuals that do not develop a tobacco relateddisease and a different pattern of amount of induction of damage ofcellular genetic material or modulation of cell homeostasis can beassociated with individuals that have developed a tobacco-relateddisease. Analysis of the amount of induction of damage of cellulargenetic material or modulation of cell homeostasis of many of suchindividuals allows the development of databases that provide an expectedtype and amount of induction of damage of cellular genetic material ormodulation of cell homeostasis that is associated or not associated witha tobacco-related disease. That is, this information can be used toprovide a baseline for an individual that is not likely to acquire atobacco-related disease (e.g., a control level exemplified bynon-tobacco users that do not develop a tobacco-related disease) and abaseline for an individual that is likely to acquire a tobacco relateddisease (e.g., a control level exemplified by tobacco users that havedeveloped a tobacco-related disease). Accordingly, when a subject isanalyzed for the predilection to develop a tobacco-related disease, theamount of induction of damage of cellular genetic material or modulationof cell homeostasis can be evaluated and, by comparing the determinedvalues to that in one or both of the databases described above, theanalyzed subject can be identified as having a predilection fordeveloping a tobacco-related disease.

Additionally, a comparison of the induction of DNA damage induced byconventional tobacco products and a tobacco product containing amodified tobacco (e.g., a genetically modified tobacco) is contemplated.By one approach, a first set of biological samples (e.g., cells of theoral cavity (cheek or gum swab) or lung cells (lung lavage)) areobtained from individuals that are consumers of conventional tobaccoproducts. These cells are analyzed for double strand DNA breaks usingone of the assays described herein. Next, the individuals are provided atobacco product comprising a modified tobacco to consume exclusively(i.e., in replacement for the conventional product). After a period oftime has passed (e.g., 1, 2, 3, or 4 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 months since the conversion from the conventional tobaccoproduct to the tobacco product containing the modified tobacco), asecond set of biological samples are taken from the individual and areanalyzed for the presence of double strand DNA breaks. It will bedetermined that fewer double strand breaks will be observed in thesecond set of biological samples than the first set, which will provideevidence that the tobacco product comprising the modified tobacco has areduced potential to contribute to a tobacco related disease (i.e., thatsaid tobacco product comprising the modified tobacco is a reduced risktobacco product).

Additionally, a reduction by a chemopreventive compound of the inductionof DNA damage induced by a tobacco product can also be measured by themethods provided herein. By one approach, a first set of biologicalsamples (e.g., cells of the oral cavity (cheek or gum swab) or lungcells (lung lavage)) are obtained from individuals that are consumers oftobacco products. These cells are analyzed for double strand DNA breaksusing one of the assays described herein. Next, the individuals areprovided a candidate chemoprotective compound to consume or use before,during, or after use of the tobacco product. After a period of time haspassed (e.g., 1, 2, 3, or 4 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months) since the commencement of using the test chemoprotectivecompound, a second set of biological samples are taken from theindividual and are analyzed for the presence of double strand DNAbreaks. It will be determined that fewer double strand breaks will beobserved in the second set of biological samples than the first set,which will provide evidence that the test chemoprotective compound canreduce the potential of tobacco to contribute to a tobacco relateddisease.

Further provided herein are kits to be used in practicing the abovemethods. In various embodiments such kits can comprise an antibody thatbinds to phosphorylated but not unphosphorylated H2AX, a reference smokeproduct, a detectably labeled second antibody that specifically binds tothe antibody that binds to phosphorylated H2AX, and suitable cells, asprovided herein elsewhere.

Also provided herein are cells containing DNA having double-strandedbreaks produced by exposure to a tobacco smoke product and, inparticular, to genetically altered cells comprising cells prepared by amethod comprising the steps of: (a) exposing a first cell population toa tobacco smoke product; (b) identifying cells containing a greaterdegree of phosphorylated H2AX relative to control cells; and (c)selectively collecting the cells identified in step (b) to form thecomposition of genetically altered cells. In preferred non-limitingembodiments, the cells having a higher degree of phosphorylated H2AX areidentified by an immunofluorescence method and selectively collected,for example by fluorescence activated cell sorting. To permit theidentification of genes associated with tobacco-induced diseases, alsoprovided herein are libraries prepared by cloning a plurality of nucleicacid molecules prepared from the cells, the cells prepared according tomethods provided for forming cells containing DNA having double-strandedbreaks produced by exposure to a tobacco smoke product, herein into aplurality of vector molecules. The following section describes severaltypes of modified tobacco that can be used with the methods describedherein.

Analysis of Changes in Cell Homeostasis: Changes in Transcriptome orProteome

High-density microarrays can be used to elucidate how cells of the oralcavity, mouth, tongue, trachea, bronchi, and lung mount a multigenicresponse to cigarette smoke and the major classes of smoke constituents(e.g., vapor and particulate phases). Using microarray technology and/orReverse Transcriptase Polymerase Chain Reaction (e.g., qRT-PCR), geneexpression patterns and levels of gene expression in short-term culturesof normal human bronchial epithelial (NHBE) cells exposed to cigarettesmoke and cigarette smoke condensates were analyzed. It was found thatsubtle alterations to the ‘homeostatic transcriptome’ are useful indefining the major signaling pathways activated upon exposure tochronic, but low level, doses of carcinogenic mixtures such as thatwhich occur daily in an individual smoker. This type of analysis isespecially relevant for complex bioactive mixtures, such as cigarettesmoke (CS), cigarette smoke condensate (CSC), tobacco smoke (TS),tobacco smoke condensate (TSC), and total particulate matter (TPM) sinceassessing the specific effects of individual components of such mixturesdoes not reflect the true impact on a cell or the body due to thesynergistic or antagonistic interactions that occur with the entirety ofthe components that are normally present. Moreover, because thecontemplated methods described herein analyze human cells of the mouth,oral cavity, trachea, and lungs, either normal or immortalized celllines (e.g., human bronchial cells (e.g., BEP2D or 16HBE140 cells),human bronchial epithelial cells (e.g., HBEC cells, 1198, or 1170-Icells), normal human bronchial epithelial cells (NHBE cells), BEAS cells(e.g., BEAS-2B), NCI-H292 cells, non-small cell lung cancer (NSCLC)cells or human alveolar cells (e.g., H460, H1792, SK-MES-1, Calu, H292,H157, H1944, H596, H522, A549, and H226) tongue cells (e.g., CAL 27),and mouth cells (e.g., Ueda-1)), which are contacted with cigarettesmoke or smoke condensates (as opposed to exposure to a single agentwith a well-defined mechanism of toxicity), one can identify uniquegenomic responses and cellular damage over time. That is, novel genesand gene expression patterns are identified using the methods describedherein because the vapor and particulate components of tobacco smokecontain numerous substances that immediately and directly damage a rangeof biomolecules, as well as, other substances whose toxicity isactivated only after biotransformation by cellular enzymes into reactivenucleophiles that then attack various cellular elements.

Although it is known that cigarette smoke, as well as various smokecomponents, can cause numerous disruptions to the genome (see Chujo etal., Lung Cancer 38: 23-29, 2002; Wistuba, et al. Semin Oncol 28: 3-13,2001), transcriptome (see Bhattacharjee, et al. Proc Natl Acad Sci USA98: 13790-13795, 2001 and Garber et al., Proc Natl Acad Sci USA 98:13784-13789, 2001), and proteome (see Hanash, et al. Dis Markers 17:295-300, 2001); relatively little is known about the effects ofcigarette smoke condensates (CSC) and cigarette smoke (CS) exposure onthe overall impact on steady state mRNA levels, transcriptionalregulation, protein production, and protein modification in normal cellsof the oral cavity, mouth, tongue, trachea, bronchi, and lung.Accordingly, experiments were conducted to identify a set of biomarkersthat could be used to monitor exposure to tobacco toxins, detectpre-malignant disease, improve diagnosis and prognosis of currenttobacco-related disease, develop patient-specific treatment options,test risk reduction strategies for current and former smokers, andidentify and develop tobacco products that have a lower potential tocontribute to a tobacco-related disease (e.g., a tobacco product thathas a lower carcinogenic potential than a conventional tobacco product,a reduced risk tobacco product). More particularly, as described herein,several approaches to identify a gene expression pattern or fingerprintfrom cells of the oral cavity, mouth, tongue, trachea, bronchi, and lung(normal or immortal), which have been exposed to tobacco smoke or atobacco smoke condensate have been discovered and the informationgenerated by practicing these methods can be used in diagnostics,therapeutic and prophylactic procedures, as well as, approaches toidentify and develop less harmful tobacco products. In addition,elucidating the various molecular, genetic, cellular, and systemiceffects of cigarette smoke provides a detailed mechanistic understandingof how chronic tobacco exposure ultimately causes disease.

Several studies assessing the clinical usefulness of alterations inglobal gene and protein expression patterns in malignant and normalhuman lung tissues have shown that quantitative and/or qualitativechanges in a small number of expressed genes and proteins, incombination with standard clinicopathological variables, have prognosticand/or diagnostic potential for patients with tobacco-related diseases.A direct cause and effect relationship between any of these documentedmolecular events and cell exposure to tobacco smoke is unclear, however.Thus, it was decided to examine the effects of tobacco constituents onthe transcriptome of normal lung cells in a controlled in vitroenvironment.

Several methods described herein analyze the transcriptome of cells ofthe oral cavity, mouth, tongue, trachea, bronchi, and lung afterexposure to a smoke or smoke condensate using high-density microarrays,qRT-PCR, or another conventional nucleic acid or protein detectionmethod such as ELISA or Western blot. The data show that exposure ofsuch cells (e.g., normal human bronchial epithelial cells (NHBE cells)to cigarette smoke or cigarette smoke condensates results in amodulation of a specific set of genes whose expression levels variedover the normal variability of gene expression in these cells.Accordingly, these genes can be used to monitor tobacco-induced changesto the transcriptome. By sorting these genes into biologicallyfunctional classes, dominant biochemical pathways known to be relevantto tobacco-related disease were identified. In addition, it wassurprising to learn that treatment with an S9 microsomal fraction, astep common in many toxicological studies, has a broad impact on geneexpression in normal lung cells that is distinctly different from theimpact of tobacco exposure.

Accordingly, some embodiments concern the identification of a gene or aplurality of genes from cells of the oral cavity, mouth, tongue,trachea, bronchi, and lung (e.g., NHBE cells), which are modulated(e.g., up-regulated or down-regulated expression) in response to contactwith a cigarette smoke (CS), a cigarette smoke condensate (CSC), tobaccosmoke (TS), tobacco smoke condensate (TSC), or total particulate matter(TPM). In some embodiments, a gene expression pattern, fingerprint, orsignature is obtained, which is an identification of a specificplurality of genes or set of genes that are modulated (i.e.,up-regulated or down-regulated) after contact with CS, CSC, TS, TSC orTPM. The plurality of genes that are affected can be any combination orsubset of genes that are identified as being influenced by exposure toCS, CSC, TS, TSC or TPM. In some embodiments, the plurality of affectedgenes are a subset of suppressor genes. In some embodiments, theplurality of genes that are affected by exposure to CS, CSC, TS, TSC orTPM are a subset of genes affecting cholesterol regulation andproduction. In some embodiments, the subset of genes that are affectedgenes are involved in oxidative stress, cell proliferation, apoptosis,protein turn-over, heat shock, ubiquitination, or endoplasmic reticulumstress.

Several approaches to conduct a gene expression analysis that involvethe use of NHBE cells are provided herein, whereby said cells arecontacted with a CS, CSC, TS, TSC or TPM and a gene, pattern of geneexpression or a fingerprint from said CS, CSC, TS, TSC or TPM-treatedcells is obtained. The gene expression data generated by the approachesdescribed herein can be recorded onto a recordable media (e.g., a harddrive, memory, cache, floppy, CD-ROM, DVD-ROM) and can be analyzed usingvarious statistical approaches to determine whether said data identifiesa genetic modulation event (e.g., an up-regulation or down-regulation ofexpression) that is statistically relevant. Statistically relevantgenetic modulation events that occur in the cells that were contactedwith a CS, CSC, TS, TSC or TPM can then be used to identify a molecularpathway that is involved in a tobacco-related disease. Accordingly, theapproaches described herein can be used to identify a marker for atobacco-related disease and to determine whether this marker ismodulated (e.g., a marker gene is up-regulated or down-regulated) inresponse to exposure to a particular CS, CSC, TS, TSC or TPM.

Furthermore, this data can be used to create a genetic profile for aparticular tobacco product, which allows one to empirically determinethe components of a given tobacco product's smoke (or tobacco per se)that contribute to a gene expression event in a human cell that isassociated with a tobacco-related disease. Accordingly, by using theapproaches described herein, one can identify specific tobacco products,as well as, growing, harvesting, curing, processing, and blendingpractices that have a reduced potential to contribute to a geneticmodulation that is associated with a tobacco-related disease. That is,the approaches described herein can be used to identify and developreduced risk cigarettes. Still further, the markers for tobacco-relateddisease, and the genetic profiles identified by using the approachesdescribed herein can be used to diagnose, provide a prognosis orotherwise identify an individual at risk of acquiring a tobacco-relateddisease and the effect of tobacco smoke on a subject at a molecularlevel. The section below describes several methods that can be used toidentify genes that are modulated after exposure to CS, CSC, TS, TSC orTPM and to identify and develop tobacco products that have a reducedrisk of contributing to a tobacco-related disease.

Tobacco Products that have a Reduced Potential to Contribute to aTobacco-Related Disease

More embodiments concern methods to identify components of a tobaccoproduct that contribute to a tobacco-related disease, the selectiveremoval or inhibition of production of these components, and thedetermination that the removal of the component(s) modulates expressionof a gene that is associated with a tobacco-related disease in a mannerthat reduces the potential for the tobacco product to contribute to atobacco related disease. It is contemplated that particular componentsof tobacco products are the factors that modulate expression of genes inhuman cells that contribute to tobacco-related disease. It is furthercontemplated that modification of genes that contribute to theproduction of these toxic components in tobacco (e.g., geneticengineering or chemical treatment) will, concomitantly, result in amodulation of gene expression in human cells that come in contact withthe smoke from said modified tobacco, which is less likely to contributeto a tobacco-related disease than the tobacco prior to modification ofthe component-producing gene. Accordingly, by selectively removing thecomponents that induce the genetic events that contribute totobacco-related disease in a human, one can develop tobacco productsthat are less likely to contribute to a tobacco-related disease.

By one approach, for example, CS is generated using a smoking machinefrom a first tobacco product that has been genetically modified to havea reduced amount of a compound. A first population of NHBE cells iscontacted with said CS obtained from the modified tobacco, as describedin Examples 4, 12, and 13. As described in these examples, the RNA isisolated and analyzed by microarray or qRT-PCR or both and a pattern ofgene expression and gene product modification events are obtained.Programs such as PathwayAssist™ software (Stratagene, La Jolla, Calif.)and/or Genespring (version 7.2, Agilent Technologies) can be used todetermine the identity of the genes that are modulated and theirrelationship to a tobacco-related disease.

A second population of NHBE cells is then contacted with CS generatedfrom the parental variety of tobacco. That is, the parental variety oftobacco is the unmodified tobacco variety used to generate the modifiedtobacco variety, wherein the unmodified tobacco retains the componentthat was removed or inhibited in the modified tobacco. As above, the RNAis isolated and analyzed by microarray or qRT-PCR or both and a patternof gene expression and gene product modification events are obtained.Programs such as PathwayAssist™ (Stratagene, La Jolla, Calif.) and/orGenespring (version 7.2, Agilent Technologies) can be used to determinethe identity of the genes that are modulated and their relationship to atobacco-related disease.

A comparison of the data obtained from the analysis of the first andsecond tobacco products will reveal that the modified tobacco modulatesfewer genes associated with a tobacco-related disease than the parental,unmodified tobacco. The data will also show that the modified tobaccoproduct induces expression of fewer proto/oncogenes. By this approach,one can effectively identify the contribution of individual componentsof a tobacco product to a tobacco-related disease. This combinatorialapproach can be used to develop tobacco products that are less likely tocontribute to a tobacco-related disease and reduced risk tobaccoproducts identified by these methods are aspects of the invention.Further, tobacco products prepared by these approaches can be preparedaccording to good manufacturing processes (GMP) (e.g., suitable for oraccepted by a governmental regulatory body, such as the Federal DrugAdministration (FDA), and containers that house said tobacco productscan comprise a label or other indicia, with or withoutstructure-function indicia, which reflects approval of said tobaccoproduct from said regulatory body. The example below describes thisapproach in greater detail.

Example 14

This example provides several approaches that can be used to obtaintobacco and tobacco products that have a reduced potential to contributeto a tobacco-related disease. Generally, these methods involve atwo-tiered analysis involving first, an analysis of a parent strain oftobacco that has a component or compound that contributes to a tobaccorelated disease and second, an analysis of a progeny of the parentstrain of tobacco that has been modified to modulate (i.e., up-regulateor down-regulate) expression of a gene that induces a cascade thatcontributes to a tobacco-related disease.

Accordingly, by one approach, a first tobacco (e.g., Burley 21 LA) thatcomprises a compound that contributes to a tobacco-related disease(e.g., nicotine) is provided. Next, preferably, smoke is obtained fromsaid first tobacco (e.g., CS), however a smoke condensate from the firsttobacco can also be obtained. Once the smoke or smoke condensate hasbeen prepared from the first tobacco, a first isolated population ofcells, preferably human cells of the mouth, tongue, trachea, bronchi, orlungs (e.g., NHBE cells) is contacted with said smoke or smokecondensate from said first tobacco. The contact can be made in a smokingchamber, for example, for less than, equal to, or more than, 5 seconds,20, seconds, 45 seconds, 1 minute, 5 minutes, 10 minutes, 15, minutes,20 minutes, 30 minutes, 45 minutes, 1 hour, two hours, three hours.Subsequent to the contact with the smoke or smoke condensate, a firstgene that is modulated (up-regulated or down-regulated) in said firstpopulation of cells in response to said contact with said smoke or smokecondensate from said first tobacco is identified (e.g., anproto/oncogene). The identification of the first gene can beaccomplished using an oligonucleotide array, microarray, qRT-PCR,nucleic acid detection (e.g., hybridization), protein detection (e.g.,antibody detection, ELISA or Western blot), or detection of a metabolite(e.g., protein fragment or cysteine) or a modified gene product (e.g.,oxidized or phosphorylated protein or amino acid). The first geneidentified as being modulated (e.g., up-regulated or down-regulated) inresponse to contact with the smoke or smoke condensate of the firsttobacco is then analyzed for its contribution to a tobacco-relateddisease. The correlation of many of the genes that are identified by theapproach above to a tobacco-related disease can be accomplished bysimply reviewing available literature or by employing commerciallyavailable software that identifies the association of a particular genewith a tobacco-related disease (e.g., PathwayAssist™ available fromStratagene, La Jolla, Calif. and/or Genespring (version 7.2, availablefrom Agilent Technologies).

Next, a second tobacco that is, preferably, the same variety and grownunder the same conditions as the first tobacco is provided. The secondtobacco has been modified to reduce expression of a second gene, a genethat contributes to the production of a compound or component present inthe first tobacco (e.g., a gene involved in nicotine synthesis, such asQPTase or PMTase). The modification of the second gene can beaccomplished by genetic engineering or chemical treatment. Severalapproaches to modify tobacco to reduce the amount of nicotine are known.(See e.g., U.S. patent application Ser. No. 10/729,121, WO0067558A1,WO9428142A1, WO05000352A1, WO05018307A1, WO03086076A1, and WO0218607A2,all of which are hereby expressly incorporated by reference in theirentireties).

By one approach, the second tobacco is genetically modified to reduceexpression of QPTase, as described above (e.g., Vector 21-41). RNAiconstructs that comprise fragments of a gene involved in nicotinesynthesis have also been used to reduce the amount of nicotine and TSNAin tobacco, as described above. By one approach, for example, the RNAiconstruct provided in FIG. 1 was used to generate a reduced nicotine andTSNA tobacco. By another approach, the RNAi construct provided in FIG. 2was used to generate a reduced nicotine and TSNA tobacco. More detailson the preparation of these RNAi constructs and the methods used tocreate transgenic tobacco having a reduced amount of nicotine and TSNAsis provided in the section that follows and Example 15.

Once the modified second tobacco is obtained, preferably a geneticallymodified second tobacco (e.g., a second tobacco that has beengenetically modified to reduce the amount of nicotine), smoke or a smokecondensate is obtained from said second tobacco. Then, a second isolatedpopulation of cells, preferably the same cell type as analyzed above(e.g., NHBE cells) is contacted with the smoke or smoke condensate fromthe second tobacco, preferably for the same amount of time as the cellsthat were contacted with the first tobacco. Subsequent to the exposureof the second population of cells to the second tobacco, an approach toidentify the modulation of gene expression in said second population ofcells is employed, preferably the same approach that was used to analyzethe first population of cells after exposure to the smoke or smokecondensate of the first tobacco product (e.g., an oligonucleotide array,microarray, qRT-PCR, nucleic acid detection (e.g., hybridization),protein detection (e.g., antibody detection, ELISA or Western blot), ordetection of a metabolite (e.g., protein fragment or cysteine) or amodified gene product (e.g., oxidized or phosphorylated protein or aminoacid).

A modulation (up-regulation or down-regulation) in expression of a firstgene that contributes to a tobacco-related disease in said secondpopulation of cells, as compared to the amount of expression of the samegene induced by the first tobacco, will be observed. This difference inexpression of a gene that is related to a tobacco-related diseaseprovides strong evidence that the modification in the second tobacco hasresulted in a tobacco that has a reduced potential to contribute to atobacco-related disease. That is, said (modified) second tobacco has areduced risk to contribute to a tobacco-related disease, as compared tothe first (unmodified) tobacco.

Conventional techniques in cultivation of said second tobacco,harvesting, curing, blending, and processing are then employed so as togenerate a tobacco product (e.g., snuff, chew, tobacco leaf, cigarette,pipe tobacco, cigar, or lozenge) and said tobacco product can beidentified as a product that has a reduced potential to contribute to atobacco-related disease as compared to a tobacco product comprising saidfirst tobacco.

It will be appreciated that the promoters used in the above-describedvectors can either be constitutive or regulatable. Constitutivepromoters are promoters that are always expressed. The constitutivepromoters selected for use in the above-described vectors can range fromweak promoters to strong promoters depending on the desired amount ofinterfering RNA to be produced. Regulatable promoters are promoters forwhich the desired level of expression can be controlled. An example of aregulatable promoter is an inducible promoter. Using an induciblepromoter in the above-described vector constructs permits expression ofa wide range of concentrations of interfering RNA inside a cell.

It will also be appreciated that there is no requirement that the sameor same types of promoters be used in vectors or multiple vector systemsthat comprise a plurality of promoters. For example, in some vectors orvector systems, a first promoter, which controls the expression of thefirst interfering RNA strand, can be an inducible promoter, whereas thesecond promoter, which controls the expression of the second RNA strand,can be a constitutive promoter. This same principal can also beillustrated in a multiple vector system. For example, a multiple vectorsystem may have three vectors each of which includes one or moredifferent types of promoters. Such a system can include, for example, afirst vector having repressible promoter that controls the expression ofan interfering RNA specific for a first gene product involved innicotine biosynthesis, a second vector having a constitutive promoterthat controls the expression of an interfering RNA specific for a secondgene product involved in nicotine biosynthesis and a third vector havingan inducible promoter that controls the expression of an interfering RNAspecific for a third gene product involved in nicotine biosynthesis.

In other embodiments, interfering RNAs can be produced synthetically andintroduced into a cell by methods known in the art. Syntheticinterfering RNAs can include a variety of RNA molecules, which include,but are not limited to, nucleic acids having at least one region ofduplex RNA. The duplex RNA in such molecules can comprise, for example,two antiparallel RNA strands that form a double-stranded RNA havingflush ends, two antiparallel RNA strands that form a double-stranded RNAhaving at least one end that forms a hairpin structure, or twoantiparallel RNA strands that form a double-stranded RNA, wherein bothends form a hairpin structure. In some embodiments, syntheticinterfering RNAs comprise a plurality of RNA duplexes.

By way of example, tobacco having reduced amounts of nicotine and TSNAsis generated from a tobacco plant that is created by exposing at leastone tobacco cell of a selected tobacco variety, such as LA Burley 21, toa nucleic acid construct comprising a promoter that is operable in aplant cell, wherein the promoter controls the expression of a RNAcomprising both strands of a duplex interfering RNA. For example, theRNA that is expressed comprises a first nucleotide sequence that issubstantially similar or identical to at least a portion of an mRNA orat least a portion of the coding strand of a gene that is involved innicotine biosynthesis. This first nucleotide sequence is followed by anon-complementary sequence that is involved in hairpin formation, andthen, a second nucleotide sequence that is complementary orsubstantially complementary to at least a portion of the firstnucleotide sequence. The exposed tobacco cell is then transformed withthe nucleic acid construct. Cells that are successfully transformed areselected using either negative selection or positive selectiontechniques and at least one tobacco plant is regenerated fromtransformed cells. The regenerated tobacco plant or portion thereof ispreferably analyzed to determine the amount of nicotine and/or TSNAspresent and these values can be compared to the amount of nicotineand/or TSNAs present in a control tobacco plant or portion thereof.Preferably the transformed and control tobacco plants are of the samevariety.

In some embodiments, a cDNA sequence encoding a plant quinolatephosphoribosyl transferase (QPTase) is used (See Example 15). As QPTaseactivity is strictly correlated with nicotine content, construction oftransgenic tobacco plants in which QPTase levels are lowered in theplant roots (compared to levels in wild-type plants) result in plantshaving reduced levels of nicotine in the leaves. Embodiments of theinvention provide methods and nucleic acid constructs for producing suchtransgenic plants, as well as, the transgenic plants themselves. Suchmethods include the expression of an interfering RNA, which lowers theamount of QPTase in tobacco roots. Other embodiments include theexpression of an interfering RNA, which lowers the amount of any QPTasethat may be present in tobacco leaves, stems and/or other tobaccotissues.

Some embodiments also concern transgenic plant cells comprising one ormore interfering RNAs that are capable of reducing or eliminating theexpression of one or more target genes and/or target gene productsinvolved in nicotine biosynthesis. As described above, an appropriateinterfering RNA comprises a duplex RNA that comprises a first strandthat is substantially similar or identical to at least a portion of atarget gene or target mRNA, which encodes a gene product involved innicotine biosynthesis. The RNA duplex also comprises a second strandthat is complementary or substantially complementary to the firststrand.

The interfering RNA or nucleic acid construct comprising the interferingRNA can be introduced into the plant cell in any suitable manner. Plantcells possessing stable interfering RNA activity, for example, by havinga nucleic acid construct stably integrated into a chromosome, can beused to regenerate whole plants using methods known in the art. As such,some aspects of the present invention relate to plants, such as tobaccoplants, transformed with one or more nucleic acid constructs and/orvectors which encode at least one interfering RNA that is capable ofreducing or eliminating the expression of a gene product involved innicotine biosynthesis. Transgenic tobacco cells and the plants describedherein are characterized in that they have a reduced amount of nicotineand/or TSNA as compared to unmodified or control tobacco cells andplants.

The tobacco plants described herein are suitable for conventionalgrowing and harvesting techniques (e.g. topping or no topping, baggingthe flowers or not bagging the flowers, cultivation in manure rich soilor without manure) and the harvested leaves and stems are suitable foruse in any traditional tobacco product including, but not limited to,pipe, cigar and cigarette tobacco and chewing tobacco in any formincluding leaf tobacco, shredded tobacco or cut tobacco. It is alsocontemplated that the low nicotine and/or TSNA tobacco described hereincan be processed and blended with conventional tobacco so as to create awide-range of tobacco products with varying amounts of nicotine and/orTSNAs. These blended tobacco products can be used in tobacco productcessation programs so as to slowly move a consumer from a high nicotineand TSNA product to a low nicotine and TSNA product. Some embodiments ofthe invention comprise a tobacco use cessation kit, comprising two ormore tobacco products with different levels of nicotine and/or TSNAs.For example, a smoker can begin the program smoking blended cigaretteshaving or delivering by FTC methodology 1-2 mg of nicotine and 0.2 μg ofTSNA, gradually move to smoking cigarettes having or delivering 0.75 mgof nicotine and 0.1 μg of TSNA, followed by cigarettes having ordelivering 0.5 mg nicotine and 0.1 μg TSNA, followed by cigaretteshaving or delivering 0.1 mg nicotine and 0.05 μg TSNA, followed bycigarettes having or delivering 0.05 mg nicotine and no detectable TSNAuntil the consumer decides to smoke only the cigarettes having virtuallyno nicotine and TSNAs or quitting smoking altogether. Accordingly, theblended cigarettes described herein provide the basis for an approach toreduce the carcinogenic potential in a human in a step-wise fashion. Thecomponents of the tobacco use cessation kit described herein may includeother tobacco products, including but not limited to, smoking materials(e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum,and lozenges.

Gene silencing has been employed in several laboratories to createtransgenic plants characterized by lower than normal amounts of specificgene products. As used herein, “exogenous” or “heterologous” nucleicacids, including DNAs and/or RNAs, refer to nucleic acids that have beenintroduced into a cell (or the cell's ancestor) through the efforts ofhumans. Such heterologous nucleic acids can be copies of a sequencewhich is naturally found in the cell being transformed, or fragmentsthereof. To produce a tobacco plant having decreased QPTase levels, anda reduced amount of nicotine and TSNAs, as compared to an untransformedor control tobacco plant or portion thereof, a tobacco cell can betransformed with an exogenous nucleic acid construct which encodes aninterfering RNA having an RNA duplex comprising a first strand that issubstantially similar or identical to at least a portion of the codingstrand of the full-length QPT cDNA sequence, a partial QPT chromosomalsequence, a full-length QPT chromosomal sequence, or an mRNA producedfrom the QPT gene. Alternatively, the tobacco cell can be transformedwith a synthetic or an in vitro transcribed interfering RNA. In someembodiments of the present invention, the interfering RNA and/or nucleicacid encoding the interfering RNA are stably transformed. In certainembodiments, the nucleic acid encoding the interfering RNA can beintegrated in the cell genome. In other embodiments, the interfering RNAand/or nucleic acid encoding the interfering RNA are transientlytransformed.

The nucleic acid constructs that are used with the transgenic plants andthe methods for producing the transgenic plants described herein encodeone or more interfering RNA constructs comprising regulatory sequences,which include, but are not limited to, a transcription initiationsequence (“promoter”) operable in the plant being transformed, and apolyadenylation/transcription termination sequence. Typically, thepromoter is located upstream of the 5′-end of the nucleotide sequence tobe expressed. The transcription termination sequence is generallylocated just downstream of the 3′-end of the nucleotide sequence to betranscribed.

In some preferred embodiments, the nucleic acid encoding the exogenousinterfering RNA, which is transformed into a tobacco cell, comprises afirst RNA strand that is identical to the an endogenous coding sequenceof a gene encoding a gene product involved in nicotine biosynthesis.However, minor variations between the exogenous and endogenous sequencescan be tolerated. It is preferred, but not necessarily required, thatthe exogenously-produced interfering RNA sequence, which issubstantially similar to the endogenous gene coding sequence, be ofsufficient similarity to the endogenous gene coding sequence, such thatthe complementary interfering RNA strand is capable of binding to theendogenous sequence in the cell to be regulated under stringentconditions as described below.

In some embodiments, the heterologous sequence utilized in the methodsof the present invention may be selected so as to produce an interferingRNA product comprising a first strand that is substantially similar oridentical to the entire QTPase mRNA sequence, or to a portion thereof,and a second strand that is complementary to the entire QPTase mRNAsequence, or to a portion thereof. The interfering RNA may becomplementary to any contiguous sequence of the natural messenger RNA.For example, it may be complementary to the endogenous mRNA sequenceproximal to the 5′-terminus or capping site, downstream from the cappingsite, between the capping site and the initiation codon and may coverall or only a portion of the non-coding region, may bridge thenon-coding and coding region, be complementary to all or part of thecoding region, complementary to the C-terminus of the coding region, orcomplementary to the 3′-untranslated region of the mRNA.

Interfering RNAs employed in carrying out the present invention includethose comprising a first strand having sequence similarity to the QPTasegene or a fragment thereof at least or equal to 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or moreconsecutive nucleotides of the QTPase. (See U.S. Pat. No. 6,586,661,which provides the sequence of the QPTase gene and protein, hereinexpressly incorporated by reference in its entirety). This definition isintended to encompass natural allelic variations in QPTase proteins.Thus, nucleic acid sequences that hybridize to nucleic acids of theQPTase gene under the conditions provided supra may also be employed incarrying out aspects of the invention. Multiple forms of the tobacco QPTenzyme may exist. Multiple forms of an enzyme may be due topost-translational modification of a single gene product, or to multipleforms of the NtQPT1 gene.

Conditions that permit other nucleic acid sequences, which code forexpression of a protein having QPTase activity, to hybridize to a QPTasegene or to other nucleic acid sequences encoding a QPTase protein can bedetermined in a routine manner. For example, hybridization of suchsequences to nucleic acids encoding the QPTase protein may be carriedout under conditions of reduced stringency or even stringent conditions(e.g., conditions represented by a wash stringency of 0.3 M NaCl, 0.03 Msodium citrate, 0.1% SDS at 60° C. or even 70° C.) herein in a standardin situ hybridization assay. See J. Sambrook et al., Molecular Cloning,A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory)). Ingeneral, such sequences will be at least 65% similar, 75% similar, 80%similar, 85% similar, 90% similar, or even 95% similar or more, with thetobacco QPTase gene, or nucleic sequences encoding the QPTase protein.Determinations of sequence similarity are made with the two sequencesaligned for maximum matching; gaps in either of the two sequences beingmatched are allowed in maximizing matching. Gap lengths of 10 or lessare preferred, gap lengths of 5 or less are more preferred, and gaplengths of 2 or less still more preferred.

Differential hybridization procedures are available which allow for theisolation of cDNA clones whose mRNA levels are as low as about 0.05% ofpoly(A)RNA. (See M. Conkling et al., Plant Physiol. 93, 1203-1211(1990)). In brief, cDNA libraries are screened using single-strandedcDNA probes of reverse transcribed mRNA from plant tissue (e.g., rootsand/or leaves). For differential screening, a nitrocellulose or nylonmembrane is soaked in 5×SSC and placed in a 96 well suction manifold;150 μL of stationary overnight culture is transferred from a masterplate to each well and vacuum applied until all liquid has passedthrough the filter. Approximately, 150 μL of denaturing solution (0.5MNaOH, 1.5 M NaCl) is placed in each well using a multiple pipetter andallowed to sit about 3 minutes. Suction is applied as above and thefilter removed and neutralized in 0.5 M Tris-HCl (pH 8.0), 1.5 M NaCl.It is then baked 2 hours in vacuo and incubated with the relevantprobes. By using nylon membrane filters and keeping master plates storedat −70° C. in 7% DMSO, filters may be screened multiple times withmultiple probes and appropriate clones recovered after several years ofstorage.

IV. Use of Tobacco Products

Nicotine Reduction and/or Tobacco-Use Cessation Programs Methods

It is also contemplated that the low nicotine and/or TSNA tobaccodescribed herein can be processed and blended with conventional tobaccoso as to create a wide-range of tobacco products with varying amounts ofnicotine and/or TSNAs. These blended tobacco products can be used innicotine reduction and/or tobacco-use cessation programs so as to move aconsumer from a high nicotine and TSNA product to a low nicotine andTSNA product.

In some embodiments provided herein, a stepwise nicotine reductionand/or tobacco-use cessation program can be established using the lownicotine, low TSNA products described above. As an example, the programparticipant initially determines his or her current level of nicotineintake. The program participant then begins the program at step 1, witha tobacco product having a reduced amount of nicotine, as compared tothe tobacco product that was used prior to beginning the program. Aftera period of time, the program participant proceeds to step 2, using atobacco product with less nicotine than the products used in step 1. Theprogram participant, after another period of time, reaches step 3,wherein the program participant begins using a tobacco product with lessnicotine than the products in step 2, and so on. Ultimately, the programparticipant uses a tobacco product having an amount of nicotine that isless than that which is sufficient to become addictive or to maintain anaddiction. Thus, the nicotine reduction and/or tobacco-use cessationprogram limits the exposure of a program participant to nicotine and,concomitantly, the harmful effect of nicotine yet retains the secondaryfactors of addiction, including but not limited to, smoke intake, oralfixation, and taste.

For example, a smoker can begin the program smoking blended cigaretteshaving or delivering 5 mg of nicotine and 1.5 μg of TSNA, gradually moveto smoking cigarettes with 3 mg of nicotine and 4 μg of TSNA, followedby cigarettes having or delivering 1 mg nicotine and 0.5 μg TSNA,followed by cigarettes having or delivering 0.5 mg nicotine and 0.25 μgTSNA, followed by cigarettes having or delivering less than 0.1 mgnicotine and less than 0.1 μg TSNA until the consumer decides to smokeonly the cigarettes having virtually no nicotine and TSNAs or quittingsmoking altogether. Preferably, a three-step program is followed wherebyat step 1, cigarettes providing 0.6 mg nicotine and less than 2 μg/gTSNA are used; at step 2, cigarettes providing 0.3 mg nicotine and lessthan 1 μg/g TSNA are used; and at step 3, cigarettes providing less than0.1 mg nicotine and less than 0.7 μg/g TSNA are used. More preferably, athree-step program is followed whereby at step 1, cigarettes providing0.6 mg nicotine and less than 2 μg/g TSNA are used; at step 2,cigarettes providing 0.3 mg nicotine and less than 1 μg/g TSNA are used;and at step 3, cigarettes providing less than 0.05 mg nicotine and lessthan 0.7 μg/g TSNA are used. Accordingly, the blended cigarettesdescribed herein provide the basis for an approach to reduce thecarcinogenic potential in a human in a step-wise fashion.

The methods described herein facilitate tobacco-use cessation byallowing the individual to retain the secondary factors of addictionsuch as smoke intake, oral fixation, and taste, while gradually reducingthe addictive nicotine levels consumed. Eventually, complete cessationis made possible because the presence of addiction for nicotine isgradually decreased while the individual is allowed to maintaindependence on the secondary factors, above.

Embodiments, for example, include stepwise blends of tobacco products,which are prepared with a variety of amounts of nicotine. These stepwiseblends are made to have reduced levels of TSNAs and varying amounts ofnicotine. As an example, cigarettes may deliver, for example, 5 mg, 4,3, 2, 1, 0.5, 0.1, or 0 mg of nicotine per cigarette. More preferably,blended cigarettes provide less than 0.01%, 0.02%, 0.03%, 0.04%, 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 0.6%nicotine.

In another aspect provided herein, the cigarettes of varying levels ofnicotine are packaged to clearly indicate the level of nicotine present,and marketed as a smoking cessation program. A preferred approach toproduce a product for nicotine reduction and/or tobacco-use cessationprogram is provided below. Individuals may wish to step up the programby skipping gradation levels of nicotine per cigarette or staying atcertain steps until ready to proceed to the next level. Significantly,embodiments provided herein allow a consumer to individually select theamount of nicotine that is ingested by selection of a particular tobaccoproduct described herein. Furthermore, because the secondary factors ofaddiction are maintained, dependence on nicotine can be reduced rapidly.

As an example, Virginia flue tobacco was blended with geneticallymodified Burley (i.e., Burley containing a significantly reduced amountof nicotine and TSNA) to yield a blended tobacco that was incorporatedinto three levels of reduced nicotine cigarettes: a step 1 cigaretteproviding 0.6 mg nicotine, a step 2 cigarette providing 0.3 mg nicotine,and a step 3 cigarette providing less than 0.05 mg nicotine. Thestepwise packs of cigarettes are clearly marked as to their nicotinecontent, and the step in the stepwise nicotine reduction program is alsoclearly marked on the package. Each week, the user purchases packscontaining cigarettes having the next lower level of nicotine, butlimits himself to no more cigarettes per day than consumed previously.The user may define his/her own rate of nicotine reduction and/orsmoking cessation according to individual needs by choosing a) thenumber of cigarettes smoked per day b) the starting nicotine levels c)the change in nicotine level per cigarette each week, and d) the finallevel of nicotine consumed per day. To keep better track of the program,the individual keeps a daily record of total nicotine intake, as well asthe number of cigarettes consumed per day. Eventually, the individualwill be consuming tobacco products with essentially no nicotine. Sincethe nicotine-free tobacco products of the final step are non-addictive,it should then be much easier to quit the use of the tobacco productsaltogether.

The nicotine reduction and/or tobacco-use cessation program limits theexposure of a program participant to nicotine while retaining thesecondary factors of addiction. These secondary factors include but arenot limited to, smoke intake, oral fixation, and taste. Because thesecondary factors are still present, the program participant may be morelikely to be successful in the nicotine reduction and/or tobacco-usecessation program than in programs that rely on supplying the programparticipant with nicotine but remove the above-mentioned secondaryfactors. Ultimately, the program participant uses a tobacco producthaving an amount of nicotine that is less than that which is sufficientto become addictive.

In another aspect provided herein, individuals would choose to obtainonly cigarettes that provide less than 0.05 mg nicotine per cigarette.Some individuals, such as individuals needing to stop nicotine intakeimmediately (for example, individuals with medical conditions orindividuals using drugs that interact with nicotine) may find thismethod useful. For some individuals, the mere presence of a cigarette inthe mouth can be enough to ease withdrawal from nicotine addiction.Gradually, the addictive properties of smoking can decrease since thereis no nicotine in the cigarettes. These individuals are then able toquit smoking entirely. More discussion on Smoking Cessation Programsthat use reduced nicotine tobacco can be found in PCT/US2004/01695,which designates the United States and was published in English, herebyexpressly incorporated by reference in its entirety.

In another aspect provided herein, packs of cigarettes containing thegradations of nicotine levels are provided as a “smoking cessation kit.”An individual who wishes to quit smoking can buy the entire kit ofcigarettes at the beginning of the program. Thus any temptation that mayoccur while buying cigarettes at the cigarette counter is avoided. Thus,the success of this method may be more likely for some individuals. Apreferred example of such a kit is provided below.

Various nicotine reduction and/or smoking cessation kits are prepared,geared to heavy, medium, or light smokers. The kits provide all of thematerials needed to quit smoking in either a two-week period (fast), aone-month period (medium) or in a two-month period (slow), depending onthe kit. Each kit contains a set number of packs of cigarettes modifiedaccording the present invention, containing either step 1 cigarettesproviding 0.6 mg nicotine, step 2 cigarettes providing 0.3 mg nicotine,and step 3 cigarettes providing less than 0.05 mg nicotine. For example,1 pack a day smokers would receive 7 packs of cigarettes, each packcontaining the above amounts of nicotine per each cigarette. Severalweeks worth of additional cigarettes provided less than 0.05 mgnicotine/cigarette would also be provided in the kit, to familiarize theconsumer with smoking no nicotine cigarettes. The kit would also containa diary for keeping track of daily nicotine intake, motivationalliterature to keep the individual interested in continuing the cessationprogram, health information on the benefits of smoking cessation, andweb site addresses to find additional anti-smoking information, such aschat groups, meetings, newsletters, recent publications, and otherpertinent links.

Some tobacco-use cessation or nicotine and/or TSNA reduction kitscomprise, for example, a conventional tobacco product and a firstreduced nicotine and/or TSNA tobacco product, wherein the first reducednicotine and/or TSNA tobacco product comprises less nicotine and/orTSNAs than the conventional tobacco product. The first reduced nicotineand/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/orTSNAs in or delivered by the first tobacco product is less than theamount of nicotine and/or TSNAs in or delivered by the conventionaltobacco product. The first reduced nicotine and/or TSNA tobacco productcan comprise treated tobacco, selectively bred low nicotine tobacco, orgenetically modified tobacco or combinations thereof. The first tobaccoproduct can also include exogenous nicotine.

Other embodiments include tobacco-use cessation or nicotine and/or TSNAreduction kits that comprise a conventional tobacco product, a firstreduced nicotine and/or TSNA tobacco product and a second reducednicotine and/or TSNA tobacco product, wherein the first reduced nicotineand/or TSNA tobacco product comprises less nicotine and/or TSNAs thanthe conventional tobacco product and the second reduced nicotine and/orTSNA tobacco product comprises less nicotine and/or TSNAs than the firstreduced nicotine and/or TSNA tobacco product. The first reduced nicotineand/or TSNA tobacco product (e.g., a cigarette) or tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g and the second reduced nicotine and/orTSNA tobacco product can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount ofnicotine and/or TSNAs in or delivered by the first tobacco product isless than the amount of nicotine and/or TSNAs in or delivered by theconventional tobacco product and the amount of nicotine and/or TSNAs inor delivered by the second tobacco product is less than the amount ofnicotine and/or TSNAs in or delivered by the first tobacco product. Thefirst and/or second reduced nicotine and/or TSNA tobacco products cancomprise treated tobacco, selectively bred low nicotine tobacco, orgenetically modified tobacco or combinations thereof. These tobaccoproducts can also include exogenous nicotine.

More embodiments include tobacco-use cessation or nicotine and/or TSNAreduction kits that comprise a conventional tobacco product, a firstreduced nicotine and/or TSNA tobacco product, a second reduced nicotineand/or TSNA tobacco product, and a third reduced nicotine and/or TSNAtobacco product, wherein the first reduced nicotine and/or TSNA tobaccoproduct comprises less nicotine and/or TSNAs than the conventionaltobacco product, the second reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the first reduced nicotineand/or TSNA tobacco product and the third reduced nicotine and/or TSNAtobacco product comprises less nicotine and/or TSNAs than the secondreduced nicotine and/or TSNA tobacco product. The first reduced nicotineand/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNAtobacco product can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the third reducednicotine and/or TSNA tobacco product can comprise (e.g., on the leaf ortobacco rod) or deliver (e.g., side-stream or main-stream smoke by theFTC and/or ISO methods), for example, less than or equal to 1.0 mg/g,0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content ofTSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g,4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long asthe amount of nicotine and/or TSNAs in or delivered by the first tobaccoproduct is less than the amount of nicotine and/or TSNAs in or deliveredby the conventional tobacco product, the amount of nicotine and/or TSNAsin or delivered by the second tobacco product is less than the amount ofnicotine and/or TSNAs in or delivered by the first tobacco product, andthe amount of nicotine and/or TSNAs in or delivered by the third tobaccoproduct is less than the amount of nicotine and/or TSNAs in or deliveredby the second tobacco product. The first, second, and/or third reducednicotine and/or TSNA tobacco products can comprise treated tobacco,selectively bred low nicotine tobacco, or genetically modified tobaccoor combinations thereof. These tobacco products can also includeexogenous nicotine.

Still more embodiments include tobacco-use cessation or nicotine and/orTSNA reduction kits that comprise a conventional tobacco product, afirst reduced nicotine and/or TSNA tobacco product, a second reducednicotine and/or TSNA tobacco product, a third reduced nicotine and/orTSNA tobacco product and a fourth reduced nicotine and/or TSNA tobaccoproduct, wherein the first reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the conventional tobaccoproduct, the second reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the first reduced nicotineand/or TSNA tobacco product, the third reduced nicotine and/or TSNAtobacco product comprises less nicotine and/or TSNAs than the secondreduced nicotine and/or TSNA tobacco product, and the fourth reducednicotine and/or TSNA tobacco product comprises less nicotine and/orTSNAs than the third reduced nicotine and/or TSNA tobacco product. Thefirst reduced nicotine and/or TSNA tobacco product (e.g., a cigarette)or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reducednicotine and/or TSNA tobacco product can comprise (e.g., on the leaf ortobacco rod) or deliver (e.g., side-stream or main-stream smoke by theFTC and/or ISO methods), for example, less than or equal to 1.0 mg/g,0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content ofTSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g,4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the thirdreduced nicotine and/or TSNA tobacco product can comprise (e.g., on theleaf or tobacco rod) or deliver (e.g., side-stream or main-stream smokeby the FTC and/or ISO methods), for example, less than or equal to 1.0mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collectivecontent of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g;and the fourth reduced nicotine and/or TSNA tobacco product can comprise(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream ormain-stream smoke by the FTC and/or ISO methods), for example, less thanor equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or acollective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less thanor equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g,or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in ordelivered by the first tobacco product is less than the amount ofnicotine and/or TSNAs in or delivered by the conventional tobaccoproduct, the amount of nicotine and/or TSNAs in or delivered by thesecond tobacco product is less than the amount of nicotine and/or TSNAsin or delivered by the first tobacco product, the amount of nicotineand/or TSNAs in or delivered by the third tobacco product is less thanthe amount of nicotine and/or TSNAs in or delivered by the secondtobacco product, and the amount of nicotine and/or TSNAs in or deliveredby the fourth tobacco product is less than the amount of nicotine and/orTSNAs in or delivered by the third tobacco product. The first, second,third, and/or fourth reduced nicotine and/or TSNA tobacco products cancomprise treated tobacco, selectively bred low nicotine tobacco, orgenetically modified tobacco or combinations thereof. These tobaccoproducts can also include exogenous nicotine.

Preferred tobacco-use cessation or nicotine and/or TSNA reduction kitscomprise, however, a first reduced nicotine and/or TSNA tobacco product,wherein the first reduced nicotine and/or TSNA tobacco product comprisesless nicotine and/or TSNAs than a conventional tobacco product. That is,in some embodiments, the tobacco-use cessation or nicotine and/or TSNAreduction kits do not contain a conventional tobacco product. The firstreduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or atobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. The first reducednicotine and/or TSNA tobacco products can comprise treated tobacco,selectively bred low nicotine tobacco, or genetically modified tobaccoor combinations thereof. The first tobacco product can also includeexogenous nicotine.

Other embodiments include tobacco-use cessation or nicotine and/or TSNAreduction kits that comprise a first reduced nicotine and/or TSNAtobacco product and a second reduced nicotine and/or TSNA tobaccoproduct, wherein the second reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the first reduced nicotineand/or TSNA tobacco product. The first reduced nicotine and/or TSNAtobacco product (e.g., a cigarette) or a tobacco therein can comprise(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream ormain-stream smoke by the FTC and/or ISO methods), for example, less thanor equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or acollective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less thanor equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g,or 0.2 μg/g and the second reduced nicotine and/or TSNA tobacco productor a tobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount ofnicotine and/or TSNAs in or delivered by the second tobacco product isless than the amount of nicotine and/or TSNAs in or delivered by thefirst tobacco product. The first and/or second reduced nicotine and/orTSNA tobacco products can comprise treated tobacco, selectively bred lownicotine tobacco, or genetically modified tobacco or combinationsthereof. These tobacco products can also include exogenous nicotine.

More embodiments include tobacco-use cessation or nicotine and/or TSNAreduction kits that comprise a first reduced nicotine and/or TSNAtobacco product, a second reduced nicotine and/or TSNA tobacco product,and a third reduced nicotine and/or TSNA tobacco product, wherein thesecond reduced nicotine and/or TSNA tobacco product comprises lessnicotine and/or TSNAs than the first reduced nicotine and/or TSNAtobacco product and the third reduced nicotine and/or TSNA tobaccoproduct comprises less nicotine and/or TSNAs than the second reducednicotine and/or TSNA tobacco product. The first reduced nicotine and/orTSNA tobacco product (e.g., a cigarette) or tobacco therein can comprise(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream ormain-stream smoke by the FTC and/or ISO methods), for example, less thanor equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or acollective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less thanor equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g,or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product ortobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the third reducednicotine and/or TSNA tobacco product or a tobacco therein can comprise(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream ormain-stream smoke by the FTC and/or ISO methods), for example, less thanor equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or acollective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less thanor equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g,or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in ordelivered by the second tobacco product is less than the amount ofnicotine and/or TSNAs in or delivered by the first tobacco product, andthe amount of nicotine and/or TSNAs in or delivered by the third tobaccoproduct is less than the amount of nicotine and/or TSNAs in or deliveredby the second tobacco product. The first, second, and/or third reducednicotine and/or TSNA tobacco products can comprise treated tobacco,selectively bred low nicotine tobacco, or genetically modified tobaccoor combinations thereof. These tobacco products can also includeexogenous nicotine.

Still more embodiments include tobacco-use cessation or nicotine and/orTSNA reduction kits that comprise a first reduced nicotine and/or TSNAtobacco product, a second reduced nicotine and/or TSNA tobacco product,a third reduced nicotine and/or TSNA tobacco product and a fourthreduced nicotine and/or TSNA tobacco product, wherein the second reducednicotine and/or TSNA tobacco product comprises less nicotine and/orTSNAs than the first reduced nicotine and/or TSNA tobacco product, thethird reduced nicotine and/or TSNA tobacco product comprises lessnicotine and/or TSNAs than the second reduced nicotine and/or TSNAtobacco product, and the fourth reduced nicotine and/or TSNA tobaccoproduct comprises less nicotine and/or TSNAs than the third reducednicotine and/or TSNA tobacco product. The first reduced nicotine and/orTSNA tobacco product (e.g., a cigarette) or a tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNAtobacco product or a tobacco therein can comprise (e.g., on the leaf ortobacco rod) or deliver (e.g., side-stream or main-stream smoke by theFTC and/or ISO methods), for example, less than or equal to 1.0 mg/g,0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content ofTSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g,4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the thirdreduced nicotine and/or TSNA tobacco product or a tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the fourth reduced nicotine and/orTSNA tobacco product or a tobacco therein can comprise (e.g., on theleaf or tobacco rod) or deliver (e.g., side-stream or main-stream smokeby the FTC and/or ISO methods), for example, less than or equal to 1.0mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collectivecontent of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/gso long as the amount of nicotine and/or TSNAs in or delivered by thesecond tobacco product is less than the amount of nicotine and/or TSNAsin or delivered by the first tobacco product, the amount of nicotineand/or TSNAs in or delivered by the third tobacco product is less thanthe amount of nicotine and/or TSNAs in or delivered by the secondtobacco product, and the amount of nicotine and/or TSNAs in or deliveredby the fourth tobacco product is less than the amount of nicotine and/orTSNAs in or delivered by the third tobacco product. The first, second,third, and/or fourth reduced nicotine and/or TSNA tobacco products cancomprise treated tobacco, selectively bred low nicotine tobacco, orgenetically modified tobacco or combinations thereof. These tobaccoproducts can also include exogenous nicotine.

The tobacco-use cessation or nicotine and/or TSNA reduction kitsdescribed herein can, optionally, comprise instructions or guidance onuse of the kit and/or tobacco-use cessation or nicotine and/or TSNAreduction and said instructions or guidance can refer the user tocounseling programs and literature on the benefits of reduced exposureto nicotine and/or TSNAs and/or tobacco products, in general. Theinstructions or guidance can be provided in said kits in the form of apaper, CD-ROM, DVD, video, cassette, website link, or other tangiblemedium. Additionally, the tobacco products provided in said tobacco-usecessation or nicotine and/or TSNA reduction kits can also compriseindicia showing that the product is a member of a series of tobaccoproducts to be consumed in a sequential order.

For example, in some embodiments, the tobacco products and/or packaginghas been labeled with a number or letter or symbol or other form ofvisually identifiable marker to indicate whether the product is aconventional tobacco product, a first tobacco product, a second tobaccoproduct, a third tobacco product, or a fourth tobacco product to be usedin said kit or otherwise in conformance with a tobacco-use cessation ornicotine and/or TSNA reduction method described herein. Preferredindicia that identifies the tobacco product as a member of a series oftobacco products used in a tobacco-use cessation or nicotine and/or TSNAreduction method include visually identifiable rings or bars that appearon the tobacco product itself and/or the tobacco product packaging (seee.g., International Publication Number WO/05041151, which designates theU.S., and was published in English, herein expressly incorporated byreference in its entirety) and Quest 1®, Quest 2®, and Quest 3®. Thetobacco-use cessation or nicotine and/or TSNA reduction kits and tobaccoproducts and packing of such can also comprise indicia from a regulatoryagency (e.g., a governmental body such as the Federal DrugAdministration) indicating that said kit or the tobacco productscontained therein have been approved for use in a tobacco-use cessationprogram.

Other embodiments concern methods of reducing the nicotine and/or TSNAconsumption or exposure of a tobacco user by providing to said tobaccouser a tobacco product or tobacco-use cessation or nicotine and/or TSNAreduction kit, as described herein. In some embodiments, a tobacco useris identified as one in need of a reduction in the consumption and/orexposure to nicotine and/or TSNAs. The identified tobacco user is thenprovided one or more of the aforementioned reduced nicotine and/or TSNAtobacco products and/or tobacco-use cessation kits described herein. Insome methods, the reduction in consumption or exposure to nicotineand/or TSNAs in said tobacco user is measured. In some methods, theabstinence from conventional tobacco use is measured.

Accordingly, by some approaches, a tobacco user, who is, optionally,identified as one in need of a reduction in the consumption or exposureto nicotine and/or TSNAs, is provided a conventional tobacco product andthen said tobacco user is provided a first reduced nicotine and/or TSNAtobacco product, wherein the first reduced nicotine and/or TSNA tobaccoproduct comprises less nicotine and/or TSNAs than the conventionaltobacco product. The first reduced nicotine and/or TSNA tobacco product(e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leafor tobacco rod) or deliver (e.g., side-stream or main-stream smoke bythe FTC and/or ISO methods), for example, less than or equal to lessthan or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotineand/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) ofless than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g,0.5 μg/g, or 0.2 μg/g. The first reduced nicotine and/or TSNA tobaccoproducts can comprise treated tobacco, selectively bred low nicotinetobacco, or genetically modified tobacco or combinations thereof. Thefirst tobacco product can also include exogenous nicotine. In somemethods, the reduction in consumption or exposure to nicotine and/orTSNAs in said tobacco user is measured. In some methods, the abstinencefrom conventional tobacco use is measured. In some methods, a marker ofnicotine addiction is measured (e.g., regional cerebral metabolic ratefor glucose and/or cerebral blood flow, which are measurable usingpositron emission tomography (PET)).

Other embodiments include tobacco-use cessation or nicotine and/or TSNAreduction methods, wherein a tobacco user, who is, optionally,identified as one in need of a reduction in the consumption or exposureto nicotine and/or TSNAs, is provided a conventional tobacco product andthen said tobacco user is provided a conventional tobacco product, afirst reduced nicotine and/or TSNA tobacco product and a second reducednicotine and/or TSNA tobacco product, wherein the first reduced nicotineand/or TSNA tobacco product comprises less nicotine and/or TSNAs thanthe conventional tobacco product and the second reduced nicotine and/orTSNA tobacco product comprises less nicotine and/or TSNAs than the firstreduced nicotine and/or TSNA tobacco product. The first reduced nicotineand/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g and the second reduced nicotine and/orTSNA tobacco product or a tobacco therein can comprise (e.g., on theleaf or tobacco rod) or deliver (e.g., side-stream or main-stream smokeby the FTC and/or ISO methods), for example, less than or equal to 1.0mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collectivecontent of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/gso long as the amount of nicotine and/or TSNAs in or delivered by thefirst tobacco product is less than the amount of nicotine and/or TSNAsin or delivered by the conventional tobacco product and the amount ofnicotine and/or TSNAs in or delivered by the second tobacco product isless than the amount of nicotine and/or TSNAs in or delivered by thefirst tobacco product. The first and/or second reduced nicotine and/orTSNA tobacco products can comprise treated tobacco, selectively bred lownicotine tobacco, or genetically modified tobacco or combinationsthereof. These tobacco products can also include exogenous nicotine. Insome methods, the reduction in consumption or exposure to nicotineand/or TSNAs in said tobacco user is measured. In some methods, theabstinence from conventional tobacco use is measured. In some methods, amarker of nicotine addiction is measured (e.g., regional cerebralmetabolic rate for glucose and/or cerebral blood flow, which aremeasurable using positron emission tomography (PET)).

More embodiments include tobacco-use cessation or nicotine and/or TSNAreduction methods, wherein a tobacco user, who is, optionally,identified as one in need of a reduction in the consumption or exposureto nicotine and/or TSNAs, is provided a conventional tobacco product, afirst reduced nicotine and/or TSNA tobacco product, a second reducednicotine and/or TSNA tobacco product, and a third reduced nicotineand/or TSNA tobacco product, wherein the first reduced nicotine and/orTSNA tobacco product comprises less nicotine and/or TSNAs than theconventional tobacco product, the second reduced nicotine and/or TSNAtobacco product comprises less nicotine and/or TSNAs than the firstreduced nicotine and/or TSNA tobacco product and the third reducednicotine and/or TSNA tobacco product comprises less nicotine and/orTSNAs than the second reduced nicotine and/or TSNA tobacco product. Thefirst reduced nicotine and/or TSNA tobacco product (e.g., a cigarette)or tobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reducednicotine and/or TSNA tobacco product or tobacco therein can comprise(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream ormain-stream smoke by the FTC and/or ISO methods), for example, less thanor equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or acollective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less thanor equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g,or 0.2 μg/g; and the third reduced nicotine and/or TSNA tobacco productor a tobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount ofnicotine and/or TSNAs in or delivered by the first tobacco product isless than the amount of nicotine and/or TSNAs in or delivered by theconventional tobacco product, the amount of nicotine and/or TSNAs in ordelivered by the second tobacco product is less than the amount ofnicotine and/or TSNAs in or delivered by the first tobacco product, andthe amount of nicotine and/or TSNAs in or delivered by the third tobaccoproduct is less than the amount of nicotine and/or TSNAs in or deliveredby the second tobacco product. The first, second, and/or third reducednicotine and/or TSNA tobacco products can comprise treated tobacco,selectively bred low nicotine tobacco, or genetically modified tobaccoor combinations thereof. These tobacco products can also includeexogenous nicotine. In some methods, the reduction in consumption orexposure to nicotine and/or TSNAs in said tobacco user is measured. Insome methods, the abstinence from conventional tobacco use is measured.In some methods, a marker of nicotine addiction is measured (e.g.,regional cerebral metabolic rate for glucose and/or cerebral blood flow,which are measurable using positron emission tomography (PET)).

Still more embodiments include tobacco-use cessation or nicotine and/orTSNA reduction methods, wherein a tobacco user, who is, optionally,identified as one in need of a reduction in the consumption or exposureto nicotine and/or TSNAs, is provided a conventional tobacco product, afirst reduced nicotine and/or TSNA tobacco product, a second reducednicotine and/or TSNA tobacco product, a third reduced nicotine and/orTSNA tobacco product and a fourth reduced nicotine and/or TSNA tobaccoproduct, wherein the first reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the conventional tobaccoproduct, the second reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the first reduced nicotineand/or TSNA tobacco product, the third reduced nicotine and/or TSNAtobacco product comprises less nicotine and/or TSNAs than the secondreduced nicotine and/or TSNA tobacco product, and the fourth reducednicotine and/or TSNA tobacco product comprises less nicotine and/orTSNAs than the third reduced nicotine and/or TSNA tobacco product. Thefirst reduced nicotine and/or TSNA tobacco product (e.g., a cigarette)or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reducednicotine and/or TSNA tobacco product or a tobacco therein can comprise(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream ormain-stream smoke by the FTC and/or ISO methods), for example, less thanor equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or acollective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less thanor equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g,or 0.2 μg/g; the third reduced nicotine and/or TSNA tobacco product or atobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the fourth reducednicotine and/or TSNA tobacco product or a tobacco therein can comprise(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream ormain-stream smoke by the FTC and/or ISO methods), for example, less thanor equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or acollective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less thanor equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g,or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in ordelivered by the first tobacco product is less than the amount ofnicotine and/or TSNAs in or delivered by the conventional tobaccoproduct, the amount of nicotine and/or TSNAs in or delivered by thesecond tobacco product is less than the amount of nicotine and/or TSNAsin or delivered by the first tobacco product, the amount of nicotineand/or TSNAs in or delivered by the third tobacco product is less thanthe amount of nicotine and/or TSNAs in or delivered by the secondtobacco product, and the amount of nicotine and/or TSNAs in or deliveredby the fourth tobacco product is less than the amount of nicotine and/orTSNAs in or delivered by the third tobacco product. The first, second,third and/or fourth reduced nicotine and/or TSNA tobacco products cancomprise treated tobacco, selectively bred low nicotine tobacco, orgenetically modified tobacco or combinations thereof. These tobaccoproducts can also include exogenous nicotine. In some methods, thereduction in consumption or exposure to nicotine and/or TSNAs in saidtobacco user is measured. In some methods, the abstinence fromconventional tobacco use is measured. In some methods, a marker ofnicotine addiction is measured (e.g., regional cerebral metabolic ratefor glucose and/or cerebral blood flow, which are measurable usingpositron emission tomography (PET)).

Preferred tobacco-use cessation or nicotine and/or TSNA reductionmethods, however, include approaches, wherein a tobacco user, who is,optionally, identified as one in need of a reduction in the consumptionor exposure to nicotine and/or TSNAs, is provided a first reducednicotine and/or TSNA tobacco product, wherein the first reduced nicotineand/or TSNA tobacco product comprises less nicotine and/or TSNAs thanthe conventional tobacco product. That is, said tobacco-use cessation ornicotine and/or TSNA reduction methods do not contain the step whereby aconventional tobacco product is provided. The first reduced nicotineand/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. The first reduced nicotine and/or TSNAtobacco products can comprise treated tobacco, selectively bred lownicotine tobacco, or genetically modified tobacco or combinationsthereof. The first tobacco product can also include exogenous nicotine.In some methods, the reduction in consumption or exposure to nicotineand/or TSNAs in said tobacco user is measured. In some methods, theabstinence from conventional tobacco use is measured. In some methods, amarker of nicotine addiction is measured (e.g., regional cerebralmetabolic rate for glucose and/or cerebral blood flow, which aremeasurable using positron emission tomography (PET)).

Other embodiments include tobacco-use cessation or nicotine and/or TSNAreduction methods, wherein a tobacco user, who is, optionally,identified as one in need of a reduction in the consumption or exposureto nicotine and/or TSNAs, is provided a first reduced nicotine and/orTSNA tobacco product and a second reduced nicotine and/or TSNA tobaccoproduct, wherein the second reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the first reduced nicotineand/or TSNA tobacco product. The first reduced nicotine and/or TSNAtobacco product (e.g., a cigarette) or a tobacco therein can comprise(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream ormain-stream smoke by the FTC and/or ISO methods), for example, less thanor equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or acollective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less thanor equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g,or 0.2 μg/g and the second reduced nicotine and/or TSNA tobacco productor a tobacco therein can comprise (e.g., on the leaf or tobacco rod) ordeliver (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount ofnicotine and/or TSNAs in or delivered by the second tobacco product isless than the amount of nicotine and/or TSNAs in or delivered by thefirst tobacco product. The first and/or second reduced nicotine and/orTSNA tobacco products can comprise treated tobacco, selectively bred lownicotine tobacco, or genetically modified tobacco or combinationsthereof. These tobacco products can also include exogenous nicotine. Insome methods, the reduction in consumption or exposure to nicotineand/or TSNAs in said tobacco user is measured. In some methods, theabstinence from conventional tobacco use is measured. In some methods, amarker of nicotine addiction is measured (e.g., regional cerebralmetabolic rate for glucose and/or cerebral blood flow, which aremeasurable using positron emission tomography (PET)).

More embodiments include tobacco-use cessation or nicotine and/or TSNAreduction methods, wherein a tobacco user, who is, optionally,identified as one in need of a reduction in the consumption or exposureto nicotine and/or TSNAs, is provided a first reduced nicotine and/orTSNA tobacco product, a second reduced nicotine and/or TSNA tobaccoproduct, and a third reduced nicotine and/or TSNA tobacco product,wherein the second reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the first reduced nicotineand/or TSNA tobacco product and the third reduced nicotine and/or TSNAtobacco product comprises less nicotine and/or TSNAs than the secondreduced nicotine and/or TSNA tobacco product. The first reduced nicotineand/or TSNA tobacco product (e.g., a cigarette) or tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNAtobacco product or tobacco therein can comprise (e.g., on the leaf ortobacco rod) or deliver (e.g., side-stream or main-stream smoke by theFTC and/or ISO methods), for example, less than or equal to 1.0 mg/g,0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content ofTSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g,4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and thethird reduced nicotine and/or TSNA tobacco product or a tobacco thereincan comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/orTSNAs in or delivered by the second tobacco product is less than theamount of nicotine and/or TSNAs in or delivered by the first tobaccoproduct, and the amount of nicotine and/or TSNAs in or delivered by thethird tobacco product is less than the amount of nicotine and/or TSNAsin or delivered by the second tobacco product. The first, second, and/orthird reduced nicotine and/or TSNA tobacco products can comprise treatedtobacco, selectively bred low nicotine tobacco, or genetically modifiedtobacco or combinations thereof. These tobacco products can also includeexogenous nicotine. In some methods, the reduction in consumption orexposure to nicotine and/or TSNAs in said tobacco user is measured. Insome methods, the abstinence from conventional tobacco use is measured.In some methods, a marker of nicotine addiction is measured (e.g.,regional cerebral metabolic rate for glucose and/or cerebral blood flow,which are measurable using positron emission tomography (PET)).

Still more embodiments include tobacco-use cessation or nicotine and/orTSNA reduction methods, wherein a tobacco user, who is, optionally,identified as one in need of a reduction in the consumption or exposureto nicotine and/or TSNAs, is provided a first reduced nicotine and/orTSNA tobacco product, a second reduced nicotine and/or TSNA tobaccoproduct, a third reduced nicotine and/or TSNA tobacco product and afourth reduced nicotine and/or TSNA tobacco product, wherein the secondreduced nicotine and/or TSNA tobacco product comprises less nicotineand/or TSNAs than the first reduced nicotine and/or TSNA tobaccoproduct, the third reduced nicotine and/or TSNA tobacco productcomprises less nicotine and/or TSNAs than the second reduced nicotineand/or TSNA tobacco product, and the fourth reduced nicotine and/or TSNAtobacco product comprises less nicotine and/or TSNAs than the thirdreduced nicotine and/or TSNA tobacco product. The first reduced nicotineand/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNAtobacco product or a tobacco therein can comprise (e.g., on the leaf ortobacco rod) or deliver (e.g., side-stream or main-stream smoke by theFTC and/or ISO methods), for example, less than or equal to 1.0 mg/g,0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content ofTSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g,4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the thirdreduced nicotine and/or TSNA tobacco product or a tobacco therein cancomprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,side-stream or main-stream smoke by the FTC and/or ISO methods), forexample, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g,1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the fourth reduced nicotine and/orTSNA tobacco product or a tobacco therein can comprise (e.g., on theleaf or tobacco rod) or deliver (e.g., side-stream or main-stream smokeby the FTC and/or ISO methods), for example, less than or equal to 1.0mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collectivecontent of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/gso long as the amount of nicotine and/or TSNAs in or delivered by thesecond tobacco product is less than the amount of nicotine and/or TSNAsin or delivered by the first tobacco product, the amount of nicotineand/or TSNAs in or delivered by the third tobacco product is less thanthe amount of nicotine and/or TSNAs in or delivered by the secondtobacco product, and the amount of nicotine and/or TSNAs in or deliveredby the fourth tobacco product is less than the amount of nicotine and/orTSNAs in or delivered by the third tobacco product. The first, second,third, and/or fourth reduced nicotine and/or TSNA tobacco products cancomprise treated tobacco, selectively bred low nicotine tobacco, orgenetically modified tobacco or combinations thereof. These tobaccoproducts can also include exogenous nicotine. In some methods, thereduction in consumption or exposure to nicotine and/or TSNAs in saidtobacco user is measured. In some methods, the abstinence fromconventional tobacco use is measured. In some methods, a marker ofnicotine addiction is measured (e.g., regional cerebral metabolic ratefor glucose and/or cerebral blood flow, which are measurable usingpositron emission tomography (PET)).

In some embodiments, the tobacco-use cessation or nicotine and/or TSNAreduction kits and tobacco use cessation methods can also comprise aconventional NRT product (e.g., nicotine patches, nicotine gum,capsules, inhalers, nasal sprays, and lozenges). That is, aspects of theinvention also include tobacco-use cessation or nicotine and/or TSNAreduction kits that comprise nicotine patches, nicotine gum, capsules,inhalers, nasal sprays, and lozenges that can be used in conjunctionwith a tobacco product as described herein. It is contemplated that theability to quit tobacco use can be increased by providing a conventionalNRT product in conjunction with one or more of the tobacco productsdescribed herein or supplementing one or more of the tobacco-usecessation methods described herein with a conventional NRT product and aconventional NRT nicotine-dependence reduction strategy. For example, atobacco-use cessation or nicotine and/or TSNA reduction program caninclude the steps of providing a tobacco user who has, optionally, beenidentified as one in need of a reduction in conventional tobacco use oneor more of the tobacco products described herein and a nicotine patch.Preferably, said tobacco user is provided a plurality of tobaccoproducts described herein and a plurality of nicotine patches, whereinat least two tobacco products and at least two nicotine patches havedifferent amounts of nicotine. That is, in some embodiments, a tobaccouser is provided a first tobacco product that comprises a tobacco thathas a reduced amount of nicotine (e.g., comprising on the leaf ortobacco rod or delivering in the side-stream or main-stream smoke, asdetermined by the FTC and/or ISO methods) less than or equal to 1.0mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g) and a nicotine patch comprisingan amount of nicotine (e.g., 21 mg, 14 mg, or 7 mg).

In some embodiments, a tobacco user is provided at least two reducednicotine tobacco products (e.g., a first tobacco product comprising onthe leaf or tobacco rod or delivering in the side-stream or main-streamsmoke, as determined by the FTC and/or ISO methods) less than or equalto 1.0 mg/g nicotine and a second tobacco product comprising (e.g., onthe leaf or tobacco rod) or delivering (e.g., side-stream or main-streamsmoke by the FTC and/or ISO methods) less than or equal to 0.6 mg/gnicotine and a nicotine patch (e.g., 21 mg, 14 mg, or 7 mg nicotine);and, in other embodiments, a tobacco user is provided at least threereduced nicotine tobacco products described herein, for example, a firsttobacco product comprising (e.g., on the leaf or tobacco rod) ordelivering (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods) less than or equal to 1.0 mg/g nicotine, a second tobaccoproduct comprising (e.g., on the leaf or tobacco rod) or delivering(e.g., side-stream or main-stream smoke by the FTC and/or ISO methods)less than or equal to 0.6 mg/g nicotine, and a third reduced nicotinetobacco product comprising (e.g., on the leaf or tobacco rod) ordelivering (e.g., side-stream or main-stream smoke by the FTC and/or ISOmethods) less than or equal to 0.3 mg/g nicotine) and a nicotine patch(e.g., 21 mg, 14 mg, or 7 mg nicotine); and, in some embodiments, atobacco user is provided at least four tobacco products describedherein, for example, a first tobacco product comprising (e.g., on theleaf or tobacco rod) or delivering (e.g., side-stream or main-streamsmoke by the FTC and/or ISO methods) less than or equal to 1.0 mg/gnicotine, a second tobacco product comprising (e.g., on the leaf ortobacco rod) or delivering (e.g., side-stream or main-stream smoke bythe FTC and/or ISO methods) less than or equal to 0.6 mg/g nicotine, athird reduced nicotine tobacco product comprising (e.g., on the leaf ortobacco rod) or delivering (e.g., side-stream or main-stream smoke bythe FTC and/or ISO methods) less than or equal to 0.3 mg/g nicotine, anda fourth reduced nicotine tobacco product comprising (e.g., on the leafor tobacco rod) or delivering (e.g., side-stream or main-stream smoke bythe FTC and/or ISO methods) less than or equal to 0.05 mg/g nicotine)and a nicotine patch (e.g., 21 mg, 14 mg, or 7 mg nicotine). Preferably,a tobacco user is provided a tobacco product that comprises (e.g., onthe leaf or tobacco rod) or delivers (e.g., side-stream or main-streamsmoke by the FTC and/or ISO methods) less than or equal to 0.05 mg/gnicotine and a nicotine patch comprising 21 mg, 14 mg, or 7 mg.

By one approach, a step 1 tobacco product is comprised of approximately25% low nicotine/TSNA tobacco and 75% conventional tobacco; a step 2tobacco product can be comprised of approximately 50% low nicotine/TSNAtobacco and 50% conventional tobacco; a step 3 tobacco product can becomprised of approximately 75% low nicotine/TSNA tobacco and 25%conventional tobacco; and a step 4 tobacco product can be comprised ofapproximately 100% low nicotine/TSNA tobacco and 0% conventionaltobacco. A tobacco-use cessation or nicotine and/or TSNA reduction kitcan comprise an amount of tobacco product from each of theaforementioned blends to satisfy a consumer for a single month program.That is, if the consumer is a one pack per day smoker, for example, asingle month kit would provide 7 packs from each step, a total of 28packs of cigarettes. Each tobacco-use cessation kit would include a setof instructions that specifically guide the consumer through thestep-by-step process. Of course, tobacco products having specificamounts of nicotine and/or TSNAs would be made available in convenientlysized amounts (e.g., boxes of cigars, packs of cigarettes, tins ofsnuff, and pouches or twists of chew) so that consumers could select theamount of nicotine and/or TSNA they individually desire. There are manyways to obtain various low nicotine/low TSNA tobacco blends using theteachings described herein and the following is intended merely to guideone of skill in the art to one possible approach.

Although the invention has been described with reference to embodimentsand examples, it should be understood that various modifications can bemade without departing from the spirit provided herein. All referencescited herein are hereby expressly incorporated by reference.

What is claimed:
 1. A method of producing a transgenic tobacco plantcomprising inhibiting expression of an endogenous gene comprising SEQ.ID. NO: 5 by RNA interference (RNAi) in a tobacco plant, wherein leavesfrom said transgenic tobacco plant have a reduced amount of nornicotineor nicotine or both nornicotine and nicotine, as compared to a controltobacco.
 2. The method of claim 1, wherein the leaves from saidtransgenic tobacco plant have a lower amount of nicotine as compared toa control tobacco.
 3. The method of claim 2, wherein the amount ofnicotine is between 2.17 mg/g and 3.99 mg/g.
 4. The method of claim 1,wherein said leaves from said transgenic tobacco plant are a tobaccoproduct.
 5. The method of claim 2, wherein said leaves from saidtransgenic tobacco plant are a tobacco product.
 6. The method of claim3, wherein said leaves from said transgenic tobacco plant are a tobaccoproduct.
 7. The method of claim 4, wherein said tobacco product is acigarette.
 8. The method of claim 5, wherein said tobacco product is acigarette.
 9. The method of claim 6, wherein said tobacco product is acigarette.
 10. The method of claim 1, wherein the amount of nornicotineis 0.00 mg/g.
 11. The method of claim 1, wherein the amount ofnornicotine is 0.06 mg/g.
 12. The method of claim 1, wherein the amountof nornicotine is 0.03 mg/g.
 13. The method of claim 2, wherein theamount of nicotine is 2.17 mg/g.
 14. The method of claim 2, wherein theamount of nicotine is 2.30 mg/g.
 15. The method of claim 2, wherein theamount of nicotine is 2.58 mg/g.
 16. The method of claim 2, wherein theamount of nicotine is 2.61 mg/g.
 17. The method of claim 2, wherein theamount of nicotine is 3.48 mg/g.
 18. The method of claim 2, wherein theamount of nicotine is 3.56 mg/g.
 19. The method of claim 2, wherein theamount of nicotine is 3.59 mg/g.
 20. The method of claim 2, wherein theamount of nicotine is 3.94 mg/g.
 21. The method of claim 2, wherein theamount of nicotine is 3.99 mg/g.